Regulation of human adenylate cyclase

ABSTRACT

Reagents which regulate human adenylate cyclase and reagents which bind to human adenylate cyclase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, peripheral and central nervous system disorders, disorders of the genito-urinary system including but not limited to benign prostatic hyperplasia and urinary incontinence, obesity, COPD and diabetes.

[0001] This application incorporates by reference U.S. provisionalapplications Serial No. 60/247,005 filed Nov. 13, 2000 and U.S.60/267,181 filed Feb. 8, 2001.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention relates to a novel human adenylate cyclase and itsregulation for therapeutic uses.

BACKGROUND OF THE INVENTION

[0003] Cyclases play important roles in the transduction ofextracellular signals via their synthesis of “secondary messengers” suchas adenosine 3′,5′-cyclic phosphate (cyclic adenosine monophosphate,cAMP) and guanosine 3′,5′-cyclic phosphate (cyclic guanosinemonophosphate, cGMP). Cell surface receptors mediate the transduction ofan extracellular signal, such as the binding of a ligand to a receptor,into a signal that is transmitted internally within the cell. Theinternal signal is carried by secondary messengers, which typically areproduced in response to the binding of an external signal. The secondarymessengers in turn activate particular proteins and other regulatorswithin the cell which have the potential to regulate expression ofspecific genes or to alter a metabolic process.

[0004] Cyclic AMP and cGMP play important roles in the regulation of amultitude of cellular activities. For example, cAM responds to cellularsignals through a specific protein kinase (cAMP-dependent protein kinaseor protein kinase A) to phosphorylate target molecules, e.g., otherprotein kinases or proteins involved in transport or cellularmorphology. Through stimulation of the kinase, intracellular cAMPmediates many of the effects of hormones in the regulation of cellularmetabolism and cell growth. Cyclic GMP also acts as an intracellularmessenger, for example, by activating cGMP-dependent kinases andregulating cGMP sensitive ion channels. The role of cGMP as a secondarymessenger has been well established in vascular smooth muscle relaxationand retinal phototransduction.

[0005] Adenylate Cyclase

[0006] The synthesis of cAMP from adenosine triphosphate (ATP) iscatalyzed by adenylate cyclase (also referred to as adenylyl cyclase andadenyl cyclase). In mammalian cells, adenylate cyclase is usually anintegral membrane protein. Adenylate cyclase activity may be affected bya factor/receptor binding event transmitted through an associated Gprotein. Interaction of several different external factors with theirdistinct receptors causes alterations in cAMP intracellularconcentration (Broach et al., U.S. Pat. No. 6,001,553). Differentreceptors are associated with their own particular G-proteinintermediary, which itself is associated with adenylate cyclase.

[0007] At least nine distinct isoenzymes of mammalian adenylate cyclasehave been identified and are designated as adenylate cyclases types 1-9(Antoni et al., U.S. Pat. No. 6,090,612). These adenylate cyclases havea general structure consisting of 12 transmembrane helices and twocytoplasmic, catalytic domains (Hurley, 1998, Curr. Opin. Struct. Biol.8:770-77). Some of these enzymes have been analyzed functionally andappear to confer unique signal processing capacities to cells (Taussiget al., 1995, J. Biol. Chem. 270:1-4).

[0008] In addition to functional diversity, adenylate cyclase isozymeshave distinct tissue distribution profiles (Iyengar, U.S. Pat. No.6,034,071). Localization studies using mRNA probes have been used todetermine tissue distribution of the various adenylate cyclases (Pieroniet al., 1993, Curr. Opin. Neurobiol. 3: 345-351). Adenylate cyclase type1 (AC 1) appears to be present only in neuronal tissue, whereas AC 2 hasbeen found in brain and lung. AC 3 has been localized in olfactoryneurons as well as other neuronal and non-neuronal tissues (Glatt andSnyder, 1993, Nature 361: 536-538; Xia et al., 1992, Neurosci. Lett.144: 169-173). AC 4 appears to be present at very low levels in brain,and throughout most tissues. AC 5 and AC 6 have also been found to bewidely distributed (Premont et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89: 9809-9813; Krupinski et al., 1992, J. Biol. Chem. 267: 24858-24862)although AC 6 seems to be of very low abundance in the brain. AC 5 isparticularly abundant in the heart and in some regions of the brain. AC7 appears to be widely distributed, but may be scarce in the brain. AC8, like AC 1, seems to be abundant in the brain. Within the brain, thedistributions of AC 1, AC 2, AC 3, AC 5 and AC 8 show distinct regionalpatterns. Collectively, these observations indicate that the particularadenylate cyclase isotype profile of a cell is fundamentally importantwith respect to cellular function.

[0009] Association with Disease

[0010] The wide variety of cyclases that have been identified thus far,together with their differing tissue distributions, demonstrate theimportance of particular cellular cyclase profiles and their roles insignal transduction with respect to cellular function. Alterations inthe levels of cyclic nucleotide intracellular (or secondary) messengerlevels that result from alterations in particular cyclase activitieshave been implicated in a wide range of conditions and diseases,including cardiovascular disease, diseases of the central nervoussystem, intestinal conditions, retinal diseases, and shock. Forinstance, the high distribution of adenylate cyclases in brain has beencorrelated with a likely role for adenylate cyclases in neurologicaldisorders such as Alzheimer's disease and Parkinson's disease. The highdistribution of adenylate cyclase in the heart is likely to contributeto cardiovascular diseases, such as angina and hypertension.

[0011] Regulation of cyclases, therefore, has important implications fortreatment of many conditions and diseases. Particular beneficialcellular responses may be elicited by blocking or stimulating theactivity of particular cyclases. The many cyclases known to date andtheir wide distribution, coupled with their specific responses to alarge number of extracellular signaling molecules, indicates that thereare likely to be many more cyclases to be identified. Thus, there is aneed in the art for identifying new cyclases and methods of regulatingcyclase activity to provide therapeutic effects.

SUMMARY OF THE INVENTION

[0012] It is an object of the invention to provide reagents and methodsof regulating a human adenylate cyclase. This and other objects of theinvention are provided by one or more of the embodiments describedbelow.

[0013] One embodiment of the invention is a adenylate cyclasepolypeptide comprising an amino acid sequence selected from the groupconsisting of:

[0014] amino acid sequences which are at least about 95% identical tothe amino acid sequence shown in SEQ ID NO: 6; and

[0015] the amino acid sequence shown in SEQ ID NO:6.

[0016] Yet another embodiment of the invention is a method of screeningfor agents which decrease extracellular matrix degradation. A testcompound is contacted with a adenylate cyclase polypeptide comprising anamino acid sequence selected from the group consisting of:

[0017] amino acid sequences which are at least about 95% identical tothe amino acid sequence shown in SEQ ID NO: 6; and

[0018] the amino acid sequence shown in SEQ ID NO:6.

[0019] Binding between the test compound and the adenylate cyclasepolypeptide is detected. A test compound which binds to the adenylatecyclase polypeptide is thereby identified as a potential agent fordecreasing extracellular matrix degradation. The agent can work bydecreasing the activity of the adenylate cyclase.

[0020] Another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a polynucleotide encoding a adenylate cyclasepolypeptide, wherein the polynucleotide comprises a nucleotide sequenceselected from the group consisting of:

[0021] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 4;

[0022] the nucleotide sequence shown in SEQ ID NO:4;

[0023] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 5; and

[0024] the nucleotide sequence shown in SEQ ID NO:5.

[0025] Binding of the test compound to the polynucleotide is detected. Atest compound which binds to the polynucleotide is identified as apotential agent for decreasing extracellular matrix degradation. Theagent can work by decreasing the amount of the adenylate cyclase throughinteracting with the adenylate cyclase mRNA.

[0026] Another embodiment of the invention is a method of screening foragents which regulate extracellular matrix degradation. A test compoundis contacted with a adenylate cyclase polypeptide comprising an aminoacid sequence selected from the group consisting of:

[0027] amino acid sequences which are at least about 95% identical tothe amino acid sequence shown in SEQ ID NO: 6; and

[0028] the amino acid sequence shown in SEQ ID NO:6.

[0029] A adenylate cyclase activity of the polypeptide is detected. Atest compound which increases adenylate cyclase activity of thepolypeptide relative to adenylate cyclase activity in the absence of thetest compound is thereby identified as a potential agent for increasingextracellular matrix degradation. A test compound which decreasesadenylate cyclase activity of the polypeptide relative to adenylatecyclase activity in the absence of the test compound is therebyidentified as a potential agent for decreasing extracellular matrixdegradation.

[0030] Even another embodiment of the invention is a method of screeningfor agents which decrease extracellular matrix degradation. A testcompound is contacted with a adenylate cyclase product of apolynucleotide which comprises a nucleotide sequence selected from thegroup consisting of:

[0031] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 4;

[0032] the nucleotide sequence shown in SEQ ID NO:4;

[0033] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 5; and

[0034] the nucleotide sequence shown in SEQ ID NO:5.

[0035] Binding of the test compound to the adenylate cyclase product isdetected. A test compound which binds to the adenylate cyclase productis thereby identified as a potential agent for decreasing extracellularmatrix degradation.

[0036] Still another embodiment of the invention is a method of reducingextracellular matrix degradation. A cell is contacted with a reagentwhich specifically binds to a polynucleotide encoding a adenylatecyclase polypeptide or the product encoded by the polynucleotide,wherein the polynucleotide comprises a nucleotide sequence selected fromthe group consisting of:

[0037] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 4;

[0038] the nucleotide sequence shown in SEQ ID NO:4;

[0039] nucleotide sequences which are at least about 50% identical tothe nucleotide sequence shown in SEQ ID NO: 5; and

[0040] the nucleotide sequence shown in SEQ ID NO:5.

[0041] Adenylate cyclase activity in the cell is thereby decreased.

[0042] Still another embodiment of the invention is a method fortreating disease. An effective amount of a pharmaceutical compositioncomprising a reagent that modulates the activity of a human adenylatecyclase or an expression vector comprising a polynucleotide encoding ahuman adenylate cyclase and a pharmaceutically acceptable carrier isadministered to a subject in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 shows the DNA-sequence (bp 1-2664 from KIAA1060) encodingthe C-terminal part of an adenylate cyclase polypeptide (SEQ ID NO:1).

[0044]FIG. 2 shows the amino acid sequence deduced from the DNA-sequenceof FIG. 1 (SEQ ID NO:3).

[0045]FIG. 3 shows a DNA-sequence encoding a novel full-length adenylatecyclase polypeptide (SEQ ID NO:4).

[0046]FIG. 4 shows the amino acid sequence deduced from the DNA-sequenceof FIG. 3 (SEQ ID NO:6).

[0047]FIG. 5 shows the genomic DNA-sequence (KIAA1060) habouring apartial CDS for the C-terminal part of an adenylate cyclase polypeptide(SEQ ID NO:2).

[0048]FIG. 6 shows the genomic DNA-sequence habouring the full CDS forthe novel adenylate cyclase polypeptide (SEQ ID NO:5).

[0049]FIG. 7 shows the amino acid sequence of the protein identified bySwissProt Accession No. P26769 (SEQ ID NO:7).

[0050]FIG. 8 shows the BLASTP alignment of human adenylate cyclase (SEQID NO:2 or 4) with the protein identified with SwissProt Accession No.P26769 (SEQ ID NO:7).

[0051]FIG. 9 shows the Prosite search results.

[0052]FIG. 10 shows the Transmembrane helices.

[0053]FIG. 11 shows the BLOCKS search results.

[0054]FIG. 12 shows the HMMPFAM—alignment of 129L_protein (SEQ ID NO:2or 4) against pfam|hmm|guanylate_cyc.

[0055]FIG. 13 shows the BLASTP—alignment of 129L_protein (SEQ ID NO:2 or4) against pdb|1CJK|1CJK-B.

[0056]FIG. 14 shows the relative expression of the novel adenylatecyclase in various tissues as determined by RT-PCR with gene specificprimers and human cDNA derived from tissue as template in a standardprocedure as known to those of skill in the art (described e.g. inEXAMPLE 6). The cDNA of the adenylate cyclase is detected in many phagelibraries reflecting the expression in a variety of different humantissues. Especially interesting is the expression in different regionsof the nervous system like brain frontal cortex and spinal cord whichemphasize the importance of adenylate cyclase in nervous systemdisorders such as primary and secondary disorders after brain injury,disorders of mood, anxiety disorders, disorders of thought and volition,disorders of sleep and wakefulness, diseases of the motor unit, such asneurogenic and myopathic disorders, neurodegenerative disorders such asAlzheimer's and Parkinson's disease, and processes of peripheral andchronic pain.

[0057]FIG. 15 shows the relative expression of the novel adenylatecyclase in various tissues as determined by RT-PCR with gene specificprimers and human phage libraries as template in a standard procedure asknown to those of skill in the art (described e.g. in EXAMPLE 6). Thefollowing tissues are represented: Lane 1-Liver, lane 2-Skeletal Muscle,lane 3-Hypothalamus, lane 4-Islets, lane 5-Adipose Sub., lane 6-AdiposeMes., lane 7-Genomic DNA, lane 8-No amplification control. Expression isshown in lanes 2-7.

[0058]FIG. 16 shows the relative expression of the novel adenylatecyclase in various tissues as determined by RT-PCR (Taqman). By far thehighest level of expression was detected in fetal and adult brain andmuscle (FIG. 16a). Detailed expression analysis reveals strongexpression in aortha (FIG. 16b) and a variety of central and peripheralCNS tissues The CNS specific expression of the adenylate cyclaseindicates the possibility to treat various disorders of the nervoussystem.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The invention relates to an isolated polynucleotide encoding aadenylate cyclase polypeptide and being selected from the groupconsisting of:

[0060] a) a polynucleotide encoding a adenylate cyclase polypeptidecomprising an amino acid sequence selected from the group consisting of:

[0061] amino acid sequences which are at least about 95% identical tothe amino acid sequence shown in SEQ ID NO: 6; and

[0062] the amino acid sequence shown in SEQ ID NO: 6.

[0063] b) a polynucleotide comprising the sequence of SEQ ID NO: 4 or 5;

[0064] c) a polynucleotide which hybridizes under stringent conditionsto a poly-nucleotide specified in (a) and (b);

[0065] d) a polynucleotide the sequence of which deviates from thepolynucleotide sequences specified in (a) to (c) due to the degenerationof the genetic code; and

[0066] e) a polynucleotide which represents a fragment, derivative orallelic variation of a polynucleotide sequence specified in (a) to (d).

[0067] Furthermore, it has been discovered by the present applicant thata novel adenylate cyclase, particularly a human adenylate cyclase, is adiscovery of the present invention. Human adenylate cyclase comprisesthe amino acid sequence shown in SEQ ID NO:6. Genomic sequences arelocated on chromosome 5. A coding sequence is shown in SEQ ID NO:4. Thegenomic sequence harbouring the CDS is shown in SEQ ID NO:5. RelatedESTs [embl|AI160340 (757 bp), embl|AI932389 (753 bp), genbank|AW967221(679 bp), embl|AA740541 (663 bp), embl|AWO55133 (613 bp), embl|N45141(614 bp), embl|AW383741 (612 bp), embl|AI095516 (513 bp), embl|AA627327(579 bp), embl|AI095516 (475 bp), embl|AI953690 (469 bp),genbank|AW964324 (576 bp), embl|AW390690 (463 bp), embl|AA192182 (447bp), embl|AWO89615 (392 bp), embl|AA194354 (472 bp), embl|AW196770 (387bp), embl|AI003215 (416 bp), embl|R12094 (477 bp), embl|T08791 (369 bp),embl|T28852 (36 lbp), embl|F22273 (430 bp), embl|R15999 (488 bp),embl|T80236 (452 bp), embl|AA224380 (393 bp), embl|T08790 (329 bp),genbank|AW901183 (348 bp), embl|H99998 (322 bp), embl|R35910 (466 bp),embl|AA913393 (304 bp), embl|R49430 (366 bp), embl|F01263 (291 bp),embl|AI703261 (278 bp), embl|AI694341 (287 bp), embl|AA913243 (256 bp),embl|AA913847 (248 bp), embl|AL120054 (384 bp)] are expressed in fetalheart; pooled fetal lung, testis and B cells; pooled kidney tumors;pooled glioblastoma; multiple sclerosis lesions; head and neck; pregnantuterus; breast; pooled germ cell tumors; stomach; skeletal muscle;pooled brain tumors; schizophrenic brain S-11 frontal lobe; infantbrain; neuroepithelial cells; melanocyte; lung carcinoid; amygdala.

[0068] Human adenylate cyclase is 95% identical over 1090 amino acids tothe rat protein identified with SwissProt Accession No. P26769 andannotated as “ADENYLATE CYCLASE, TYPE II (EC 4.6.1.1)” (FIG. 45).

[0069] Human adenylate cyclase of the invention is expected to be usefulfor the same purposes as previously identified adenylate cyclaseenzymes. Human adenylate cyclase is believed to be useful in therapeuticmethods to treat disorders such as peripheral and central nervous systemdisorders, disorders of the genito-urinary system including but notlimited to benign prostatic hyperplasia and urinary incontinence,obesity, COPD and diabetes. Human adenylate cyclase also can be used toscreen for human adenylate cyclase activators and inhibitors.

[0070] Polypeptides

[0071] Human adenylate cyclase polypeptides according to the inventioncomprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, or 1086 contiguous amino acids selected from SEQ IDNO:6 or a biologically active variant thereof, as defined below. Aadenylate cyclase polypeptide of the invention therefore can be aportion of a adenylate cyclase protein, a full-length adenylate cyclaseprotein, or a fusion protein comprising all or a portion of a adenylatecyclase protein.

[0072] Biologically Active Variants

[0073] Human adenylate cyclase polypeptide variants which arebiologically active, e.g., retain an adenylate cyclase activity, alsoare adenylate cyclase polypeptides. Preferably, naturally ornon-naturally occurring adenylate cyclase polypeptide variants haveamino acid sequences which are at least about 95, 96, 96, or 98%identical to the amino acid sequence shown in SEQ ID NO:6 or a fragmentthereof. Percent identity between a putative adenylate cyclasepolypeptide variant and an amino acid sequence of SEQ ID NO:6 isdetermined by conventional methods. See, for example, Altschul et al.,Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1992). Briefly, two amino acidsequences arealigned to optimize the alignment scores using a gap opening penalty of10, a gap extension penalty of, l, and the “BLOSUM62” scoring matrix ofHenikoff and Henikoff (ibid.). Those skilled in the art appreciate thatthere are many established algorithms available to align two amino acidsequences. The “FASTA” similarity search algorithm of Pearson and Lipmanis a suitable protein alignment method for examining the level ofidentity shared by an amino acid sequence disclosed herein and the aminoacid sequence of a putative variant. The FASTA algorithm is described byPearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444(1988), and byPearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA firstcharacterizes sequence similarity by identifying regions shared by thequery sequence (e.g. SEQ ID NO: 6) and a test sequence that have eitherthe highest density of identities (if the ktup variable is 1) or pairsof identities (if ktup=2), without considering conservative aminoacidsubstitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using anamino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gapopeningpenalty=10, gap extension penalty=1, andsubstitution matrix=BLOSUM62. These parameters can be introduced into aFASTA program by modifying the scoring matrix file (“SMATRIX”), asexplained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990). FASTAcan also be used to determine the sequence identity of nucleic acidmolecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

[0074] Variations in percent identity can be due, for example, to aminoacid substitutions, insertions, or deletions. Amino acid substitutionsare defined as one for one amino acid replacements. They areconservative in nature when the substituted amino acid has similarstructural and/or chemical properties. Examples of conservativereplacements are substitution of a leucine with an isoleucine or valine,an aspartate with a glutamate, or a threonine with a serine.

[0075] Amino acid insertions or deletions are changes to or within anamino acid sequence. They typically fall in the range of about 1 to 5amino acids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of a adenylate cyclase polypeptide can be foundusing computer programs well known in the art, such as DNASTAR software.Whether an amino acid change results in a biologically active adenylatecyclase polypeptide can readily be determined by assaying for adenylatecyclase activity, as described for example, in U.S. Pat. No. 5,795,756.

[0076] Fusion Proteins

[0077] Fusion proteins are useful for generating antibodies againstadenylate cyclase polypeptide amino acid sequences and for use invarious assay systems. For example, fusion proteins can be used toidentify proteins which interact with portions of a adenylate cyclasepolypeptide. Protein affinity chromatography or library-based assays forprotein-protein interactions, such as the yeast two-hybrid or phagedisplay systems, can be used for this purpose. Such methods are wellknown in the art and also can be used as drug screens.

[0078] A adenylate cyclase polypeptide fusion protein comprises twopolypeptide segments fused together by means of a peptide bond. Thefirst polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, or 887 contiguous amino acids selected from SEQ IDNO:3 or at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200,250, 300, 350; 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, or 1086 contiguous amino acids selected from SEQ IDNO:6 or of a biologically active variant, such as those described above.The first polypeptide segment also can comprise full-length adenylatecyclase protein.

[0079] The second polypeptide segment can be a full-length protein or aprotein fragment. Proteins commonly used in fusion protein constructioninclude β-galactosidase, β-glucuronidase, green fluorescent protein(GFP), auto fluorescent proteins, including blue fluorescent protein(BFP), glutathione-S-transferase (GST), luciferase, horseradishperoxidase (HRP), and chloramphenicol acetyltransferase (CAT).Additionally, epitope tags are used in fusion protein constructions,including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusionconstructions can include maltose binding protein (MBP), S-tag, Lex aDNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. A fusion protein alsocan be engineered to contain a cleavage site located between theadenylate cyclase polypeptide-encoding sequence and the heterologousprotein sequence, so that the adenylate cyclase polypeptide can becleaved and purified away from the heterologous moiety.

[0080] A fusion protein can be synthesized chemically, as is known inthe art. Preferably, a fusion protein is produced by covalently linkingtwo polypeptide segments or by standard procedures in the art ofmolecular biology. Recombinant DNA methods can be used to prepare fusionproteins, for example, by making a DNA construct which comprises codingsequences selected from the complement of SEQ ID NO:4, or 5 in properreading frame with nucleotides encoding the second polypeptide segmentand expressing the DNA construct in a host cell, as is known in the art.Many kits for constructing fusion proteins are available from companiessuch as Promega Corporation (Madison, Wis.), Stratagene (La Jolla,Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology(Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown,Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0081] Identification of Species Homologs

[0082] Species homologs of human adenylate cyclase polypeptide can beobtained using adenylate cyclase polypeptide polynucleotides (describedbelow) to make suitable probes or primers for screening cDNA expressionlibraries from other species, such as mice, monkeys, or yeast,identifying cDNAs which encode homologs of adenylate cyclasepolypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

[0083] A adenylate cyclase polynucleotide can be single- ordouble-stranded and comprises a coding sequence or the complement of acoding sequence for a adenylate cyclase polypeptide. Coding sequencesfor human adenylate cyclase are shown in SEQ ID NOS: 4 and 5.

[0084] Degenerate nucleotide sequences encoding human adenylate cyclasepolypeptides, as well as homologous nucleotide sequences which are atleast about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98%identical to the nucleotide sequence shown in SEQ ID NOS:4 and 5 ortheir complements also are adenylate cyclase polynucleotides. Percentsequence identity between the sequences of two polynucleotides isdetermined using computer programs such as ALIGN which employ the FASTAalgorithm, using an affine gap search with a gap open penalty of −12 anda gap extension penalty of −2. Complementary DNA (cDNA) molecules,species homologs, and variants of adenylate cyclase polynucleotideswhich encode biologically active adenylate cyclase polypeptides also areadenylate cyclase polynucleotides. Polynucleotide fragments comprising8, 10, 12, 15, 18, 20, 25, 50, 75, 100, 200, 300, 400, or 500 contiguousnucleotides selected from SEQ ID NO:4 or 5 or their complements also areadenylate cyclase polynucleotides.

[0085] Identification of Polynucleotide Variants and Homologs

[0086] Variants and homologs of the adenylate cyclase polynucleotidesdescribed above also are adenylate cyclase polynucleotides. Typically,homologous adenylate cyclase polynucleotide sequences can be identifiedby hybridization of candidate polynucleotides to known adenylate cyclasepolynucleotides under stringent conditions, as is known in the art. Forexample, using the following wash conditions—2×SSC (0.3 M NaCl, 0.03 Msodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minuteseach; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, roomtemperature twice, 10 minutes each—homologous sequences can beidentified which contain at most about 25-30% basepair mismatches. Morepreferably, homologous nucleic acid strands contain 15-25% basepairmismatches, even more preferably 5-15% basepair mismatches.

[0087] Species homologs of the adenylate cyclase polynucleotidesdisclosed herein also can be identified by making suitable probes orprimers and screening cDNA expression libraries from other species, suchas mice, monkeys, or yeast. Human variants of adenylate cyclasepolynucleotides can be identified, for example, by screening human cDNAexpression libraries. It is well known that the T_(m) of adouble-stranded DNA decreases by 1-1.5° C. with every 1% decrease inhomology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of humanadenylate cyclase polynucleotides or adenylate cyclase polynucleotidesof other species can therefore be identified by hybridizing a putativehomologous adenylate cyclase polynucleotide with a polynucleotide havinga nucleotide sequence of SEQ ID NO: 4 or 5 or the complement thereof toform a test hybrid. The melting temperature of the test hybrid iscompared with the melting temperature of a hybrid comprisingpolynucleotides having perfectly complementary nucleotide sequences, andthe number or percent of basepair mismatches within the test hybrid iscalculated.

[0088] Nucleotide sequences which hybridize to adenylate cyclasepolynucleotides or their complements following stringent hybridizationand/or wash conditions also are adenylate cyclase polynucleotides.Stringent wash conditions are well known and understood in the art andare disclosed, for example, in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

[0089] Typically, for stringent hybridization conditions a combinationof temperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a adenylate cyclase polynucleotidehaving a nucleotide sequence shown in SEQ ID NO:4 or 5 or the complementthereof and a polynucleotide sequence which is at least about 50,preferably about 75, 90, 96, or 98% identical to one of those nucleotidesequences can be calculated, for example, using the equation of Boltonand McCarthy, Proc. Natl. Acad. Sci USA. 48, 1390 (1962):

T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)−600/l),where l=the length of the hybrid in basepairs.

[0090] Stringent wash conditions include, for example, 4×SSC at 65° C.,or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

[0091] Preparation of Polynucleotides

[0092] A adenylate cyclase polynucleotide can be isolated free of othercellular components such as membrane components, proteins, and lipids.Polynucleotides can be made by a cell and isolated using standardnucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated adenylate cyclasepolynucleotides. For example, restriction enzymes and probes can be usedto isolate polynucleotide fragments which comprises adenylate cyclasenucleotide sequences. Isolated polynucleotides are in preparations whichare free or at least 70, 80, or 90% free of other molecules.

[0093] Human adenylate cyclase cDNA molecules can be made with standardmolecular biology techniques, using adenylate cyclase mRNA as atemplate. Human adenylate cyclase cDNA molecules can thereafter bereplicated using molecular biology techniques known in the art anddisclosed in manuals such as Sambrook et al. (1989). An amplificationtechnique, such as PCR, can be used to obtain additional copies ofpolynucleotides of the invention, using either human genomic DNA or cDNAas a template.

[0094] Alternatively, synthetic chemistry techniques can be used tosynthesizes adenylate cyclase polynucleotides. The degeneracy of thegenetic code allows alternate nucleotide sequences to be synthesizedwhich will en code a adenylate cyclase polypeptide having, for example,an amino acid sequence shown in SEQ ID NO: 6 or a biologically activevariant thereof.

Extending Polynucleotides

[0095] Various PCR-based methods can be used to extend the nucleic acidsequences disclosed herein to detect upstream sequences such aspromoters and regulatory elements. For example, restriction-site PCRuses universal primers to retrieve unknown sequence adjacent to a knownlocus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA isfirst amplified in the presence of a primer to a linker sequence and aprimer specific to the known region. The amplified sequences are thensubjected to a second round of PCR with the same linker primer andanother specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase andsequenced using reverse transcriptase.

[0096] Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16, 8186, 1988). Primers can be designed using commerciallyavailable software, such as OLIGO 4.06 Primer Analysis software(National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0097] Another method which can be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom et al., PCR MethodsApplic. 1, 111-119, 1991). In this method, multiple restriction enzymedigestions and ligations also can be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR

[0098] Another method which can be used to retrieve unknown sequences isthat of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991).Additionally, PCR, nested primers, and PROMOTERFINDER libraries(CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH,Palo Alto, Calif.). This process avoids the need to screen libraries andis useful in finding intron/exon junctions.

[0099] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs.Randomly-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariescan be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0100] Commercially available capillary electrophoresis systems can beused to analyze the size or confirm the nucleotide sequence of PCR orsequencing products. For example, capillary sequencing can employflowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity can be converted to electrical signalusing appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR,Perkin Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display can be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

Obtaining Polypeptides

[0101] Human adenylate cyclase polypeptides can be obtained, forexample, by purification from human cells, by expression of adenylatecyclase polynucleotides, or by direct chemical synthesis.

[0102] Protein Purification

[0103] Human adenylate cyclase polypeptides can be purified from anycell which expresses the enzyme, including host cells which have beentransfected with adenylate cyclase expression constructs. A purifiedadenylate cyclase polypeptide is separated from other compounds whichnormally associate with the adenylate cyclase polypeptide in the cell,such as certain proteins, carbohydrates, or lipids, using methodswell-known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified adenylate cyclasepolypeptides is at least 80% pure; preferably, the preparations are 90%,95%, or 99% pure. Purity of the preparations can be assessed by anymeans known in the art, such as SDS-polyacrylamide gel electrophoresis.

[0104] Expression of Polynucleotides

[0105] To express a adenylate cyclase polynucleotide, the polynucleotidecan be inserted into an expression vector which contains the necessaryelements for the transcription and translation of the inserted codingsequence. Methods which are well known to those skilled in the art canbe used to construct expression vectors containing sequences encodingadenylate cyclase polypeptides and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrooket al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

[0106] A variety of expression vector/host systems can be utilized tocontain and express sequences encoding a adenylate cyclase polypeptide.These include, but are not limited to, microorganisms, such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors,insect cell systems infected with virus expression vectors (e.g.,baculovirus), plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids),or animal cell systems.

[0107] The control elements or regulatory sequences are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like canbe used. The baculovirus polyhedrin promoter can be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO, and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) can be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of a nucleotide sequenceencoding a adenylate cyclase polypeptide, vectors based on SV40 or EBVcan be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

[0108] In bacterial systems, a number of expression vectors can beselected depending upon the use intended for the adenylate cyclasepolypeptide. For example, when a large quantity of a adenylate cyclasepolypeptide is needed for the induction of antibodies, vectors whichdirect high level expression of fusion proteins that are readilypurified can be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding theadenylate cyclase polypeptide can be ligated into the vector in framewith sequences for the amino-terminal Met and the subsequent 7 residuesof β-galactosidase so that a hybrid protein is produced. pIN vectors(Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEXvectors (Promega, Madison, Wis.) also can be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems can be designed to include heparin, thrombin, or factor Xaprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0109] In the yeast Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al.(1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

[0110] If plant expression vectors are used, the expression of sequencesencoding adenylate cyclase polypeptides can be driven by any of a numberof promoters. For example, viral promoters such as the ³⁵S and 19Spromoters of CaMV can be used alone or in combination, with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680,1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., ResultsProbl. Cell Differ. 17, 85-105, 1991). These constructs can beintroduced into plant cells by direct DNA transformation or bypathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (e.g., Hobbs or Murray, in McGRAWHILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y.,pp. 191-196, 1992).

[0111] An insect system also can be used to express a adenylate cyclasepolypeptide. For example, in one such system Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.Sequences encoding adenylate cyclase polypeptides can be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofadenylate cyclase polypeptides will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses can then be used to infect S. frugiperda cells or Trichoplusialarvae in which adenylate cyclase polypeptides can be expressed(Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

[0112] A number of viral-based expression systems can be used to expressadenylate cyclase polypeptides in mammalian host cells. For example, ifan adenovirus is used as an expression vector, sequences encodingadenylate cyclase polypeptides can be ligated into an adenovirustranscription/translation complex comprising the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome can be used to obtain a viable virus which iscapable of expressing a adenylate cyclase polypeptide in infected hostcells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). Ifdesired, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, can be used to increase expression in mammalian host cells.

[0113] Human artificial chromosomes (HACs) also can be used to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6M to 10M are constructed and delivered to cells viaconventional delivery methods (e.g., liposomes, polycationic aminopolymers, or vesicles).

[0114] Specific initiation signals also can be used to achieve moreefficient translation of sequences encoding adenylate cyclasepolypeptides. Such signals include the ATG initiation codon and adjacentsequences. In cases where sequences encoding a adenylate cyclasepolypeptide, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals (including the ATG initiation codon)should be provided. The initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons can be of various origins,both natural and synthetic. The efficiency of expression can be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used (see Scharf et al., Results Probl. CellDiffer. 20, 125-162, 1994).

[0115] Host Cells

[0116] A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedadenylate cyclase polypeptide in the desired fashion. Such modificationsof the polypeptide include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. Post-translational processing which cleaves a “prepro” formof the polypeptide also can be used to facilitate correct insertion,folding and/or function. Different host cells which have specificcellular machinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC; 10801 University Boulevard,Manassas, Va. 20110-2209) and can be chosen to ensure the correctmodification and processing of the foreign protein.

[0117] Stable expression is preferred for long-term, high-yieldproduction of recombinant proteins. For example, cell lines which stablyexpress adenylate cyclase polypeptides can be transformed usingexpression vectors which can contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellscan be allowed to grow for 1-2 days in an enriched medium before theyare switched to a selective medium. The purpose of the selectable markeris to confer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced adenylatecyclase sequences. Resistant clones of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltype. See, for example, ANIMAL CELL CULTURE, R. I. Freshney, ed., 1986.

[0118] Any number of selection systems can be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32,1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22,817-23, 1980) genes which can be employed in tk⁻ or aprf⁻ cells,respectively. Also, antimetabolite, antibiotic, or herbicide resistancecan be used as the basis for selection. For example, dhfr confersresistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77,3567-70, 1980), npt confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), andals and pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murray, 1992, supra). Additionalselectable genes have been described. For example, trpB allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, can be used to identify transformants and to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131,1995).

[0119] Detecting Expression

[0120] Although the presence of marker gene expression suggests that theadenylate cyclase polynucleotide is also present, its presence andexpression may need to be conformed. For example, if a sequence encodinga adenylate cyclase polypeptide is inserted within a marker genesequence, transformed cells containing sequences which encode aadenylate cyclase polypeptide can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding a adenylate cyclase polypeptide under the control ofa single promoter. Expression of the marker gene in response toinduction or selection usually indicates expression of the adenylatecyclase polynucleotide.

[0121] Alternatively, host cells which contain a adenylate cyclasepolynucleotide and which express a adenylate cyclase polypeptide can beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassay or immunoassay techniqueswhich include membrane, solution, or chip-based technologies for thedetection and/or quantification of nucleic acid or protein. For example,the presence of a polynucleotide sequence encoding a adenylate cyclasepolypeptide can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes or fragments or fragments of polynucleotidesencoding a adenylate cyclase polypeptide. Nucleic acidamplification-based assays involve the use of oligonucleotides selectedfrom sequences encoding a adenylate cyclase polypeptide to detecttransformants which contain a adenylate cyclase polynucleotide.

[0122] A variety of protocols for detecting and measuring the expressionof a adenylate cyclase polypeptide, using either polyclonal ormonoclonal antibodies specific for the polypeptide, are known in theart. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay using monoclonal antibodiesreactive to two non-interfering epitopes on a adenylate cyclasepolypeptide can be used, or a competitive binding assay can be employed.These and other assays are described in Hampton et al., SEROLOGICALMETHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) andMaddox et al., J. Exp. Med 158, 1211-1216, 1983).

[0123] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingadenylate cyclase polypeptides include oligolabeling, nick translation,end-labeling, or PCR amplification using a labeled nucleotide.Alternatively, sequences encoding a adenylate cyclase polypeptide can becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and can be used tosynthesize RNA probes in vitro by addition of labeled nucleotides and anappropriate RNA polymerase such as T7, T3, or SP6. These procedures canbe conducted using a variety of commercially available kits (AmershamPharmacia Biotech, Promega, and US Biochemical). Suitable reportermolecules or labels which can be used for ease of detection includeradionuclides, enzymes, and fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0124] Expression and Purification of Polypeptides

[0125] Host cells transformed with nucleotide sequences encoding aadenylate cyclase polypeptide can be cultured under conditions suitablefor the expression and recovery of the protein from cell culture. Thepolypeptide produced by a transformed cell can be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode adenylate cyclase polypeptidescan be designed to contain signal sequences which direct secretion ofsoluble adenylate cyclase polypeptides through a prokaryotic oreukaryotic cell membrane or which direct the membrane insertion ofmembrane-bound adenylate cyclase polypeptide.

[0126] As discussed above, other constructions can be used to join asequence encoding a adenylate cyclase polypeptide to a nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Inclusion ofcleavable linker sequences such as those specific for Factor Xa orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the adenylate cyclase polypeptide also can be used tofacilitate purification. One such expression vector provides forexpression of a fusion protein containing a adenylate cyclasepolypeptide and 6 histidine residues preceding a thioredoxin or anenterokinase cleavage site. The histidine residues facilitatepurification by IMAC (immobilized metal ion affinity chromatography, asdescribed in Porath et al., Prot. Exp. Purif 3, 263-281, 1992), whilethe enterokinase cleavage site provides a means for purifying theadenylate cyclase polypeptide from the fusion protein. Vectors whichcontain fusion proteins are disclosed in Kroll et al., DNA Cell Biol.12, 441-453, 1993.

[0127] Chemical Synthesis

[0128] Sequences encoding a adenylate cyclase polypeptide can besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223,1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a adenylate cyclase polypeptide itself can be producedusing chemical methods to synthesize its amino acid sequence, such as bydirect peptide synthesis using solid-phase techniques (Merrifield, J.Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269,202-204, 1995). Protein synthesis can be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of adenylate cyclase polypeptides can beseparately synthesized and combined using chemical methods to produce afull-length molecule.

[0129] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, W H Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic adenylate cyclasepolypeptide can be confirmed by amino acid analysis or sequencing (e.g.,the Edman degradation procedure; see Creighton, supra). Additionally,any portion of the amino acid sequence of the adenylate cyclasepolypeptide can be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins to produce a variantpolypeptide or a fusion protein.

[0130] Production of Altered Polypeptides

[0131] As will be understood by those of skill in the art, it may beadvantageous to produce adenylate cyclase polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producean RNA transcript having desirable properties, such as a half-life whichis longer than that of a transcript generated from the naturallyoccurring sequence.

[0132] The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter adenylate cyclasepolypeptide-encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the polypeptide or mRNA product. DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides can be used to engineer the nucleotide sequences. Forexample, site-directed mutagenesis can be used to insert new restrictionsites, alter glycosylation patterns, change codon preference, producesplice variants, introduce mutations, and so forth.

Antibodies

[0133] Any type of antibody known in the art can be generated to bindspecifically to an epitope of a adenylate cyclase polypeptide.“Antibody” as used herein includes intact immunoglobulin molecules, aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding an epitope of a adenylate cyclase polypeptide.Typically, at least 6, 8, 10, or 12 contiguous amino acids are requiredto form an epitope. However, epitopes which involve non-contiguous aminoacids may require more, e.g., at least 15, 25, or 50 amino acids.

[0134] An antibody which specifically binds to an epitope of a adenylatecyclase polypeptide can be used therapeutically, as well as inimmunochemical assays, such as Western blots, ELISAs, radioimmunoassays,immunohistochemical assays, immunoprecipitations, or otherimmunochemical assays known in the art. Various immunoassays can be usedto identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody whichspecifically binds to the immunogen.

[0135] Typically, an antibody which specifically binds to a adenylatecyclase polypeptide provides a detection signal at least 5-, 10-, or20-fold higher than a detection signal provided with other proteins whenused in an immunochemical assay. Preferably, antibodies whichspecifically bind to adenylate cyclase polypeptides do not detect otherproteins in immunochemical assays and can immunoprecipitate a adenylatecyclase polypeptide from solution.

[0136] Human adenylate cyclase polypeptides can be used to immunize amammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, toproduce polyclonal antibodies. If desired, a adenylate cyclasepolypeptide can be conjugated to a carrier protein, such as bovine serumalbumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on thehost species, various adjuvants can be used to increase theimmunological response. Such adjuvants include, but are not limited to,Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surfaceactive substances (e.g. lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially useful.

[0137] Monoclonal antibodies which specifically bind to a adenylatecyclase polypeptide can be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These techniques include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J.Immunol. Methods 81, 3142, 1985; Cote et al., Proc. Natl. Acad. Sci 80,2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

[0138] In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.Alternatively, humanized antibodies can be produced using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto a adenylate cyclase polypeptide can contain antigen binding siteswhich are either partially or fully humanized, as disclosed in U.S. Pat.No. 5,565,332.

[0139] Alternatively, techniques described for the production of singlechain antibodies can be adapted using methods known in the art toproduce single chain antibodies which specifically bind to adenylatecyclase polypeptides. Antibodies with related specificity, but ofdistinct idiotypic composition, can be generated by chain shuffling fromrandom combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad.Sci. 88, 11120-23, 1991).

[0140] Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-111). Single-chainantibodies can be mono- or bispecific, and can be bivalent- ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

[0141] A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth 165,81-91).

[0142] Antibodies which specifically bind to adenylate cyclasepolypeptides also can be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter etal., Nature 349, 293-299, 1991).

[0143] Other types of antibodies can be constructed and usedtherapeutically in methods of the invention. For example, chimericantibodies can be constructed as disclosed in WO 93/03151. Bindingproteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared.

[0144] Antibodies according to the invention can be purified by methodswell known in the art. For example, antibodies can be affinity purifiedby passage over a column to which a adenylate cyclase polypeptide isbound. The bound antibodies can then be eluted from the column using abuffer with a high salt concentration.

Antisense Oligonucleotides

[0145] Antisense oligonucleotides are nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofadenylate cyclase gene products in the cell.

[0146] Antisense oligonucleotides can be deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages suchalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, Meth Mol. Biol. 20, 1-8, 1994; Sonveaux,Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583,1990.

[0147] Modifications of adenylate cyclase gene expression can beobtained by designing antisense oligonucleotides which will formduplexes to the control, 5′, or regulatory regions of the adenylatecyclase gene. Oligonucleotides derived from the transcription initiationsite, e.g., between positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using “triple helix”base-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orchaperons. Therapeutic advances using triplex DNA have been described inthe literature (e.g., Gee et al., in Huber & Carr, MOLECULAR ANDIMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994).An antisense oligonucleotide also can be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0148] Precise complementarity is not required for successful complexformation between an antisense oligonucleotide and the complementarysequence of a adenylate cyclase polynucleotide. Antisenseoligonucleotides which comprise, for example, 2, 3, 4, or 5 or morestretches of contiguous nucleotides which are precisely complementary toa adenylate cyclase polynucleotide, each separated by a stretch ofcontiguous nucleotides which are not complementary to adjacent adenylatecyclase nucleotides, can provide sufficient targeting specificity foradenylate cyclase mRNA. Preferably, each stretch of complementarycontiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotidesin length. Non-coplementary intervening sequences are preferably 1, 2,3, or 4 nucleotides in length. One skilled in the art can easily use thecalculated melting point of an antisense-sense pair to determine thedegree of mismatching which will be tolerated between a particularantisense oligonucleotide and a particular adenylate cyclasepolynucleotide sequence.

[0149] Antisense oligonucleotides can be modified without affectingtheir ability to hybridize to a adenylate cyclase polynucleotide. Thesemodifications can be internal or at one or both ends of the antisensemolecule. For example, internucleoside phosphate linkages can bemodified by adding cholesteryl or diamine moieties with varying numbersof carbon residues between the amino groups and terminal ribose.Modified bases and/or sugars, such as arabinose instead of ribose, or a3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the5′ phosphate group are substituted, also can be employed in a modifiedantisense oligonucleotide. These modified oligonucleotides can beprepared by methods well known in the art. See, e.g., Agrawal et al.,Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90,543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

[0150] Ribozymes are RNA molecules with catalytic activity. See, e.g.,Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59,543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture& Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

[0151] The coding sequence of a adenylate cyclase polynucleotide can beused to generate ribozymes which will specifically bind to mRNAtranscribed from the adenylate cyclase polynucleotide. Methods ofdesigning and constructing ribozymes which can cleave other RNAmolecules in trans in a highly sequence specific manner have beendeveloped and described in the art (see Haseloff et al. Nature 334,585-591, 1988). For example, the cleavage activity of ribozymes can betargeted to specific RNAs by engineering a discrete “hybridization”region into the ribozyme. The hybridization region contains a sequencecomplementary to the target RNA and thus specifically hybridizes withthe target (see, for example, Gerlach et al., EP 321,201).

[0152] Specific ribozyme cleavage sites within a adenylate cyclase RNAtarget can be identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target RNA containingthe cleavage site can be evaluated for secondary structural featureswhich may render the target inoperable. Suitability of candidateadenylate cyclase RNA targets also can be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays. Longer complementary sequences can beused to increase the affinity of the hybridization sequence for thetarget. The hybridizing and cleavage regions of the ribozyme can beintegrally related such that upon hybridizing to the target RNA throughthe complementary regions, the catalytic region of the ribozyme cancleave the target.

[0153] Ribozymes can be introduced into cells as part of a DNAconstruct. Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease adenylate cyclase expression.Alternatively, if it is desired that the cells stably retain the DNAconstruct, the construct can be supplied on a plasmid and maintained asa separate element or integrated into the genome of the cells, as isknown in the art. A ribozyme-encoding DNA construct can includetranscriptional regulatory elements, such as a promoter element, anenhancer or UAS element, and a transcriptional terminator signal, forcontrolling transcription of ribozymes in the cells.

[0154] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymescan be engineered so that ribozyme expression will occur in response tofactors which induce expression of a target gene. Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of mRNA occurs only when both a ribozyme and a target geneare induced in the cells.

Differentially Expressed Genes

[0155] Described herein are methods for the identification of geneswhose products interact with human adenylate cyclase. Such genes mayrepresent genes which are differentially expressed in disordersincluding, but not limited to, peripheral and central nervous systemdisorders, disorders of the genito-urinary system including but notlimited to benign prostatic hyperplasia and urinary incontinence,obesity, COPD and diabetes. Further, such genes may represent geneswhich are differentially regulated in response to manipulations relevantto the progression or treatment of such diseases. Additionally, suchgenes may have a temporally modulated expression, increased or decreasedat different stages of tissue or organism development. A differentiallyexpressed gene may also have its expression modulated under controlversus experimental conditions. In addition, the human adenylate cyclasegene or gene product may itself be tested for differential expression.

[0156] The degree to which expression differs in a normal versus adiseased state need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

[0157] Identification of Differentially Expressed Genes

[0158] To identify differentially expressed genes total RNA or,preferably, mRNA is isolated from tissues of interest. For example, RNAsamples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquewhich does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Ausubel et al.,ed.,, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.New York, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

[0159] Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes are identified by methodswell known to those of skill in the art. They include, for example,differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85,208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308,149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and,preferably, differential display (Liang & Pardee, Science 257, 967-71,1992; U.S. Pat. No. 5,262,311).

[0160] The differential expression information may itself suggestrelevant methods for the treatment of disorders involving the humanadenylate cyclase. For example, treatment may include a modulation ofexpression of the differentially expressed genes and/or the geneencoding the human adenylate cyclase. The differential expressioninformation may indicate whether the expression or activity of thedifferentially expressed gene or gene product or the human adenylatecyclase gene or gene product are up-regulated or down-regulated.

Screening Methods

[0161] The invention provides assays for screening test compounds whichbind to or modulate the activity of a adenylate cyclase polypeptide or aadenylate cyclase polynucleotide. A test compound preferably binds to aadenylate cyclase polypeptide or polynucleotide. More preferably, a testcompound decreases or increases adenylate cyclase activity by at leastabout 10, preferably about 50, more preferably about 75, 90, or 100%relative to the absence of the test compound.

[0162] Test Compounds

[0163] Test compounds can be pharmacologic agents already known in theart or can be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

[0164] Methods for the synthesis of molecular libraries are well knownin the art (see, for example, DeWitt et al., Proc. Natl. Acad Sci U.S.A.90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci U.S.A. 91, 11422, 1994;Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science261, 1303, 1993; Carell et al., Angew. Chem. Int; Ed. Engl. 33, 2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop etal., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can bepresented in solution (see, e.g., Houghten, BioTechniques 13, 412-421,1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No.5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89,1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990;Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad.Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; andLadner, U.S. Pat. No. 5,223,409).

[0165] High Throughput Screening

[0166] Test compounds can be screened for the ability to bind toadenylate cyclase polypeptides or polynucleotides or to affect adenylatecyclase activity or adenylate cyclase gene expression using highthroughput screening. Using high throughput screening, many discretecompounds can be tested in parallel so that large numbers of testcompounds can be quickly screened. The most widely establishedtechniques utilize 96-well microtiter plates. The wells of themicrotiter plates typically require assay volumes that range from 50 to500 μl. In addition to the plates, many instruments, materials,pipettors, robotics, plate washers, and plate readers are commerciallyavailable to fit the 96-well format.

[0167] Alternatively, “free format assays,” or assays that have nophysical barrier between samples, can be used. For example, an assayusing pigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

[0168] Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light. Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

[0169] Yet another example is described by Salmon et al., MolecularDiversity 2, 57-63 (1996). In this example, combinatorial libraries werescreened for compounds that had cytotoxic effects on cancer cellsgrowing in agar.

[0170] Another high throughput screening method is described in Beutelet al., U.S. Pat. No. 5,976,813. In this method, test samples are placedin a porous matrix. One or more assay components are then placed within,on top of, or at the bottom of a matrix such as a gel, a plastic sheet,a filter, or other form of easily manipulated solid support. Whensamples are introduced to the porous matrix they diffuse sufficientlyslowly, such that the assays can be performed without the test samplesrunning together.

[0171] Binding Assays

[0172] For binding assays, the test compound is preferably a smallmolecule which binds to and occupies, for example, the active site ofthe adenylate cyclase polypeptide, such that normal biological activityis prevented. Examples of such small molecules include, but are notlimited to, small peptides or peptide-like molecules.

[0173] In binding assays, either the test compound or the adenylatecyclase polypeptide can comprise a detectable label, such as afluorescent, radioisotopic, chemiluminescent, or enzymatic label, suchas horseradish peroxidase, alkaline phosphatase, or luciferase.Detection of a test compound which is bound to the adenylate cyclasepolypeptide can then be accomplished, for example, by direct counting ofradioemmission, by scintillation counting, or by determining conversionof an appropriate substrate to a detectable product.

[0174] Alternatively, binding of a test compound to a adenylate cyclasepolypeptide can be determined without labeling either of theinteractants. For example, a microphysiometer can be used to detectbinding of a test compound with a adenylate cyclase polypeptide. Amicrophysiometer (e.g., Cytosensor™) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a test compound and a adenylate cyclase polypeptide (McConnellet al., Science 257, 1906-1912, 1992).

[0175] Determining the ability of a test compound to bind to a adenylatecyclase polypeptide also can be accomplished using a technology such asreal-time Bimolecular Interaction Analysis (BIA) (Sjolander &Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al., Curr.Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore™). Changes in the optical phenomenon surfaceplasmon resonance (SPR) can be used as an indication of real-timereactions between biological molecules.

[0176] In yet another aspect of the invention, a adenylate cyclasepolypeptide can be used as a “bait protein” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.,Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268, 12046-12054,1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al.,Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify otherproteins which bind to or interact with the adenylate cyclasepolypeptide and modulate its activity.

[0177] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding aadenylate cyclase polypeptide can be fused to a polynucleotide encodingthe DNA binding domain of a known transcription factor (e.g., GAL4). Inthe other construct a DNA sequence that encodes an unidentified protein(“prey” or “sample”) can be fused to a polynucleotide that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact in vivo to form anprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ), which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detected,and cell colonies containing the functional transcription factor can beisolated and used to obtain the DNA sequence encoding the protein whichinteracts with the adenylate cyclase polypeptide.

[0178] It may be desirable to immobilize either the adenylate cyclasepolypeptide (or polynucleotide) or the test compound to facilitateseparation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the adenylate cyclase polypeptide (or polynucleotide) or the testcompound can be bound to a solid support. Suitable solid supportsinclude, but are not limited to, glass or plastic slides, tissue cultureplates, microtiter wells, tubes, silicon chips, or particles such asbeads (including, but not limited to, latex, polystyrene, or glassbeads). Any method known in the art can be used to attach the enzymepolypeptide (or polynucleotide) or test compound to a solid support,including use of covalent and non-covalent linkages, passive absorption,or pairs of binding moieties attached respectively to the polypeptide(or polynucleotide) or test compound and the solid support. Testcompounds are preferably bound to the solid support in an array, so thatthe location of individual test compounds can be tracked. Binding of atest compound to a adenylate cyclase polypeptide (or polynucleotide) canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicrocentrifuge tubes.

[0179] In one embodiment, the adenylate cyclase polypeptide is a fusionprotein comprising a domain that allows the adenylate cyclasepolypeptide to be bound to a solid support. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed adenylatecyclase polypeptide; the mixture is then incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components. Binding of the interactantscan be determined either directly or indirectly, as described above.Alternatively, the complexes can be dissociated from the solid supportbefore binding is determined.

[0180] Other techniques for immobilizing proteins or polynucleotides ona solid support also can be used in the screening assays of theinvention. For example, either a adenylate cyclase polypeptide (orpolynucleotide) or a test compound can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated adenylate cyclasepolypeptides (or polynucleotides) or test compounds can be prepared frombiotin-NHS(N-hydroxysuccinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies which specifically bind to aadenylate cyclase polypeptide, polynucleotide, or a test compound, butwhich do not interfere with a desired binding site, such as the activesite of the adenylate cyclase polypeptide, can be derivatized to thewells of the plate. Unbound target or protein can be trapped in thewells by antibody conjugation.

[0181] Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies which specifically bind tothe adenylate cyclase polypeptide or test compound, enzyme-linked assayswhich rely on detecting an activity of the adenylate cyclasepolypeptide, and SDS gel electrophoresis under non-reducing conditions.

[0182] Screening for test compounds which bind to a adenylate cyclasepolypeptide or polynucleotide also can be carried out in an intact cell.Any cell which comprises a adenylate cyclase polypeptide orpolynucleotide can be used in a cell-based assay system. A adenylatecyclase polynucleotide can be naturally occurring in the cell or can beintroduced using techniques such as those described above. Binding ofthe test compound to a adenylate cyclase polypeptide or polynucleotideis determined as described above.

[0183] Enzyme Assays

[0184] Test compounds can be tested for the ability to increase ordecrease the adenylate cyclase activity of a human adenylate cyclasepolypeptide. Adenylate cyclase activity can be measured, for example, asdescribed in U.S. Pat. No. 5,795,756.

[0185] Enzyme assays can be carried out after contacting either apurified adenylate cyclase polypeptide, a cell membrane preparation, oran intact cell with a test compound. A test compound which decreasesenzyme activity of a adenylate cyclase polypeptide by at least about 10,preferably about 50, more preferably about 75, 90, or 100% is identifiedas a potential therapeutic agent for decreasing adenylate cyclaseactivity. A test compound which increases enzyme activity of a humanadenylate cyclase polypeptide by at least about 10, preferably about 50,more preferably about 75, 90, or 100% is identified as a potentialtherapeutic agent for increasing human adenylate cyclase activity.

[0186] Gene Expression

[0187] In another embodiment, test compounds which increase or decreaseadenylate cyclase gene expression are identified. A adenylate cyclasepolynucleotide is contacted with a test compound, and the expression ofan RNA or polypeptide product of the adenylate cyclase polynucleotide isdetermined. The level of expression of appropriate mRNA or polypeptidein the presence of the test compound is compared to the level ofexpression of mRNA or polypeptide in the absence of the test compound.The test compound can then be identified as a modulator of expressionbased on this comparison. For example, when expression of mRNA orpolypeptide is greater in the presence of the test compound than in itsabsence, the test compound is identified as a stimulator or enhancer ofthe mRNA or polypeptide expression. Alternatively, when expression ofthe mRNA or polypeptide is less in the presence of the test compoundthan in its absence, the test compound is identified as an inhibitor ofthe mRNA or polypeptide expression.

[0188] The level of adenylate cyclase mRNA or polypeptide expression inthe cells can be determined by methods well known in the art fordetecting mRNA or polypeptide. Either qualitative or quantitativemethods can be used. The presence of polypeptide products of a adenylatecyclase polynucleotide can be determined, for example, using a varietyof techniques known in the art, including immunochemical methods such asradioimmunoassay, Western blotting, and immunohistochemistry.Alternatively, polypeptide synthesis can be determined in vivo, in acell culture, or in an in vitro translation system by detectingincorporation of labeled amino acids into a adenylate cyclasepolypeptide.

[0189] Such screening can be carried out either in a cell-free assaysystem or in an intact cell. Any cell which expresses a adenylatecyclase polynucleotide can be used in a cell-based assay system. Theadenylate cyclase polynucleotide can be naturally occurring in the cellor can be introduced using techniques such as those described above.Either a primary culture or an established cell line, such as CHO orhuman embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

[0190] The invention also provides pharmaceutical compositions which canbe administered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise, for example,a adenylate cyclase polypeptide, adenylate cyclase polynucleotide,ribozymes or antisense oligonucleotides, antibodies which specificallybind to a adenylate cyclase polypeptide, or mimetics, activators, orinhibitors of a adenylate cyclase polypeptide activity. The compositionscan be administered alone or in combination with at least one otheragent, such as stabilizing compound, which can be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions can be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

[0191] In addition to the active ingredients, these pharmaceuticalcompositions can contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0192] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof such as sodiumalginate.

[0193] Dragee cores can be used in conjunction with suitable coatings,such as concentrated sugar solutions, which also can contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, ie., dosage.

[0194] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0195] Pharmaceutical formulations suitable for parenteraladministration can be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions can contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds can beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers also can be used for delivery.Optionally, the suspension also can contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. For topical or nasaladministration, penetrants appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0196] The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

[0197] Further details on techniques for formulation and administrationcan be found in the latest edition of REMINGTON'S PHARMACEUTICALSCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labeled for treatment of an indicated condition Suchlabeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

[0198] The human adenylate cyclase of the invention can be regulated totreat peripheral and central nervous system disorders, disorders of thegenito-urinary system including but not limited to benign prostatichyperplasia and urinary incontinence, obesity, COPD and diabetes.

[0199] Peripheral and Central Nervous System Disorders.

[0200] Peripheral and central nervous system disorders which may betreated include brain injuries, cerebrovascular diseases and theirconsequences, Parkinson's disease, corticobasal degeneration, motorneuron disease, dementia, including ALS, multiple sclerosis, traumaticbrain injury, stroke, post-stroke, post-traumatic brain injury, andsmall-vessel cerebrovascular disease. Dementias, such as Alzheimer'sdisease, vascular dementia, dementia with Lewy bodies, frontotemporaldementia and Parkinsonism linked to chromosome 17, frontotemporaldementias, including Pick's disease, progressive nuclear palsy,corticobasal degeneration, Huntington's disease, thalamic degeneration,Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia,and Korsakoffs psychosis also can be treated. Similarly, it may bepossible to treat cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders,attention deficit hyperactivity disorders, and memory disturbances inchildren with learning disabilities, by regulating the activity of humanadenyl cyclase.

[0201] Pain that is associated with peripheral and central nervoussystem disorders also can be treated by regulating the activity of humanadenyl cyclase. Pain which can be treated includes that associated withcentral nervous system disorders, such as multiple sclerosis, spinalcord injury, sciatica, failed back surgery syndrome, traumatic braininjury, epilepsy, Parkinson's disease, post-stroke, and vascular lesionsin the brain and spinal cord (e.g., infarct, hemorrhage, vascularmalformation). Non-central neuropathic pain includes that associatedwith post mastectomy pain, reflex sympathetic dystrophy (RSD),trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS relatedpain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neuralgias, and post-herpeticneuralgia. Pain associated with cancer and cancer treatment also can betreated, as can headache pain (for example, migraine with aura, migrainewithout aura, and other migraine disorders), episodic and chronictension-type headache, tension-type like headache, cluster headache, andchronic paroxysmal hemicrania

[0202] Obesity.

[0203] Obesity and overweight are defined as an excess of body fatrelative to lean body mass. An increase in caloric intake or a decreasein energy expenditure or both can bring about this imbalance leading tosurplus energy being stored as fat. Obesity is associated with importantmedical morbidities and an increase in mortality. The causes of obesityare poorly understood and may be due to genetic factors, environmentalfactors or a combination of the two to cause a positive energy balance.In contrast, anorexia and cachexia are characterized by an imbalance inenergy intake versus energy expenditure leading to a negative energybalance and weight loss. Agents that either increase energy expenditureand/or decrease energy intake, absorption or storage would be usefullfor treating obesity, overweight, and associated comorbidities. Agentsthat either increase energy intake and/or decrease energy expenditure orincrease the amount of lean tissue would be useful for treatingcachexia, anorexia and wasting disorders.

[0204] This gene, translated proteins and agents which modulate thisgene or portions of the gene or its products are useful for treatingobesity, overweight, anorexia, cachexia, wasting disorders, appetitesuppression, appetite enhancement, increases or decreases in satiety,modulation of body weight, and/or other eating disorders such asbulimia. Also this gene, translated proteins and agents which modulatethis gene or portions of the gene or its products are useful fortreating obesity/overweight-associated comorbidities includinghypertension, type 2 diabetes, coronary artery disease, hyperlipidemia,stroke, gallbladder disease, gout, osteoarthritis, sleep apnea andrespiratory problems, some types of cancer including endometrial,breast, prostate, and colon cancer, thrombolic disease, polycysticovarian syndrome, reduced fertility, complications of pregnancy,menstrual irregularities, hirsutism, stress incontinence, anddepression.

[0205] Urinary Incontinence

[0206] Urinary incontinence (UI) is the involuntary loss of urine. Urgeurinary incontinence (UUI) is one of the most common types of UItogether with stress urinary incontinence (SUI) which is usually causedby a defect in the urethral closure mechanism. UUI is often associatedwith neurological disorders or diseases causing neuronal damages such asdementia, Parkinson's disease, multiple sclerosis, stroke and diabetes,although it also occurs in individuals with no such disorders. One ofthe usual causes of UUI is overactive bladder (OAB) which is a medicalcondition referring to the symptoms of frequency and urgency derivedfrom abnormal contractions and instability of the detrusor muscle.

[0207] There are several medications for urinary incontinence on themarket today mainly to help treating UUI. Therapy for OAB is focused ondrugs that affect peripheral neural control mechanisms or those that actdirectly on bladder detrusor smooth muscle contraction, with a majoremphasis on development of anticholinergic agents. These agents caninhibit the parasympathetic nerves which control bladder voiding or canexert a direct spasmolytic effect on the detrusor muscle of the bladder.This results in a decrease in intravesicular pressure, an increase incapacity and a reduction in the frequency of bladder contraction. Orallyactive anticholinergic drugs such as propantheline (ProBanthine),tolterodine tartrate (Detrol) and oxybutynin (Ditropan) are the mostcommonly prescribed drugs. However, their most serious drawbacks areunacceptable side effects such as dry mouth, abnormal visions,constipation, and central nervous system disturbances. These sideeffects lead to poor compliance. Dry mouth symptoms alone areresponsible for a 70% non-compliance rate with oxybutynin. Theinadequacies of present therapies highlight the need for novel,efficacious, safe, orally available drugs that have fewer side effects.

[0208] Benign Prostatic Hyperplacia

[0209] Benign prostatic hyperplacia (BPH) is the benign nodularhyperplasia of the periuretlral prostate gland commonly seen in men overthe age of 50. The overgrowth occurs in the central area of the prostatecalled the transition zone, which wraps around the urethra BPH causesvariable degrees of bladder outlet obstruction resulting in progressivelower urinary tract syndromes (LUTS) characterized by urinary frequency,urgency, and nocturia due to incomplete emptying and rapid refilling ofthe bladder. The actual cause of BPH is unknown but may involveage-related alterations in balance of steroidal sex hormones.

[0210] The selective ??1-adrenoceptor antagonists, such as prazosin,indoramin and tamsulosin are used as an adjunct in the symptomatictreatment of urinary obstruction caused by BPH, although they do notaffect on the underlying cause of BPH. In BPH, increased sympathetictone exacerbates the degree of obstruction of the urethra throughcontraction of prostatic and urethral smooth muscle. These compoundsinhibit sympathetic activity, thereby relaxing the smooth muscle of theurinary tract. Uroselective ??1-antagonists and ??1-antagonists withhigh tissue selectivity for lower urinary tract smooth muscle that donot provoke hypotensive side-effects should be developed for thetreatment.

[0211] Drugs blocking dihydrotestosterone have been used to reduce thesize of the prostate. 5??-reductase inhibitors such as finasteride areprescribed for BPH. These agents selectively inhibit 5??-reductase whichmediates conversion of testosterone to dihydrotestosterone, therebyreducing plasma dihydrotestosterone levels and thus prostate growth. The5??-reductase inhibitors do not bind to androgen receptors and do notaffect testosterone levels nor do they possess feminizing side-effects.

[0212] Androgen receptor antagonists are used for the treatment ofprostatic hyperplasia due to excessive action or production oftestosterone. Various antiandrogens are under investigation for BPHincluding chlormadione derivatives with no estrogenic activity,orally-active aromatase inhibitors, luteinizing hormone-releasinghormone (LHRH) analogues.

[0213] COPD

[0214] Chronic obstructive pulmonary (or airways) disease (COPD) is acondition defined physiologically as airflow obstruction that generallyresults from a mixture of emphysema and peripheral airway obstructiondue to chronic bronchitis (Senior & Shapiro, Pulmonary Diseases andDisorders, 3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998;Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized bydestruction of alveolar walls leading to abnormal enlargement of the airspaces of the lung. Chronic bronchitis is defined clinically as thepresence of chronic productive cough for three months in each of twosuccessive years. In COPD, airflow obstruction is usually progressiveand is only partially reversible. By far the most important risk factorfor development of COPD is cigarette smoking, although the disease doesoccur in non-smokers.

[0215] Chronic inflammation of the airways is a key pathological featureof COPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages which are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

[0216] Diabetes

[0217] Diabetes mellitus is a common metabolic disorder characterized byan abnormal elevation in blood glucose, alterations in lipids andabnormalities (complications) in the cardiovascular system, eye, kidneyand nervous system. Diabetes is divided into two separate diseases: type1 diabetes (juvenile onset), which results from a loss of cells whichmake and secrete insulin, and type 2 diabetes (adult onset), which iscaused by a defect in insulin secretion and a defect in insulin action.

[0218] Type 1 diabetes is initiated by an autoimuune reaction thatattacks the insulin secreting cells (beta cells) in the pancreaticislets. Agents that prevent this reaction from occurring or that stopthe reaction before destruction of the beta cells has been accomplishedare potential therapies for this disease. Other agents that induce betacell proliferation and regeneration also are potential therapies.

[0219] Type II diabetes is the most common of the two diabeticconditions (6% of the population). The defect in insulin secretion is animportant cause of the diabetic condition and results from an inabilityof the beta cell to properly detect and respond to rises in bloodglucose levels with insulin release. Therapies that increase theresponse by the beta cell to glucose would offer an important newtreatment for this disease.

[0220] The defect in insulin action in Type II diabetic subjects isanother target for therapeutic intervention. Agents that increase theactivity of the insulin receptor in muscle, liver, and fat will cause adecrease in blood glucose and a normalization of plasma lipids. Thereceptor activity can be increased by agents that directly stimulate thereceptor or that increase the intracellular signals from the receptor.Other therapies can directly activate the cellular end process, i.e.glucose transport or various enzyme systems, to generate an insulin-likeeffect and therefore a produce beneficial outcome. Because overweightsubjects have a greater susceptibility to Type II diabetes, any agentthat reduces body weight is a possible therapy.

[0221] Both Type I and Type diabetes can be treated with agents thatmimic insulin action or that treat diabetic complications by reducingblood glucose levels. Likewise, agents that reduces new blood vesselgrowth can be used to treat the eye complications that develop in bothdiseases.

[0222] This invention further pertains to the use of novel agentsidentified by the screening assays described above. Accordingly, it iswithin the scope of this invention to use a test compound identified asdescribed herein in an appropriate animal model. For example, an agentidentified as described herein (e.g., a modulating agent, an antisensenucleic acid molecule, a specific antibody, ribozyme, or a adenylatecyclase polypeptide binding molecule) can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0223] A reagent which affects adenylate cyclase activity can beadministered to a human cell, either in vitro or in vivo, to reduceadenylate cyclase activity. The reagent preferably binds to anexpression product of a human adenylate cyclase gene. If the expressionproduct is a protein, the reagent is preferably an antibody. Fortreatment of human cells ex vivo, an antibody can be added to apreparation of stem cells which have been removed from the body. Thecells can then be replaced in the same or another human body, with orwithout clonal propagation, as is known in the art.

[0224] In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung, liver, spleen, heart brain, lymph nodes, and skin.

[0225] A liposome useful in the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of thetargeted cell to deliver its contents to the cell. Preferably, thetransfection efficiency of a liposome is about 0.5 μg of DNA per 16mmole of liposome delivered to about 10⁶ cells, more preferably about1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, andeven more preferably about 2.0 μg of DNA per 16 nmol of liposomedelivered to about 10 ⁶ cells. Preferably, a liposome is between about100 and 500 nm, more preferably between about 150 and 450 nm, and evenmore preferably between about. 200 and 400 nm in diameter.

[0226] Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to aparticular cell type, such as a cell-specific ligand exposed on theouter surface of the liposome.

[0227] Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods which arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 nmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 nmol liposomes.

[0228] In another embodiment, antibodies can be delivered to specifictissues in vivo using receptor-mediated targeted delivery.Receptor-mediated DNA delivery techniques are taught in, for example,Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al.,GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu etal., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad.Sci. U.S.A. 87; 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 33842(1991).

[0229] Determination of a Therapeutically Effective Dose

[0230] The determination of a therapeutically effective dose is Wellwithin the capability of those skilled in the art. A therapeuticallyeffective dose refers to that amount of active ingredient whichincreases or decreases adenylate cyclase activity relative to theadenylate cyclase activity which occurs in the absence of thetherapeutically effective dose.

[0231] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays or in animal models,usually mice, rabbits, dogs, or pigs. The animal model also can be usedto determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

[0232] Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population), can be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀.

[0233] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0234] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

[0235] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0236] If the reagent is a single-chain antibody, polynucleotidesencoding the antibody can be constructed and introduced into a celleither ex vivo or in vivo using well-established techniques including,but not limited to, transferrin-polycation-mediated DNA transfer,transfection with naked or encapsulated nucleic acids, liposome-mediatedcellular fusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, “gene gun,” andDEAE- or calcium phosphate-mediated transfection.

[0237] Effective in vivo dosages of an antibody are in the range ofabout 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μgto about 500 μg/kg of patient body weight, and about 200 to about 250μg/kg of patient body weight. For administration of polynucleotidesencoding single-chain antibodies, effective in vivo dosages are in therange of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μgto about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100μg of DNA.

[0238] If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides which expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

[0239] Preferably, a reagent reduces expression of a adenylate cyclasegene or the activity of a adenylate cyclase polypeptide by at leastabout 10, preferably about 50, more preferably about 75, 90, or 100%relative to the absence of the reagent. The effectiveness of themechanism chosen to decrease the level of expression of a adenylatecyclase gene or the activity of a adenylate cyclase polypeptide can beassessed using methods well known in the art, such as hybridization ofnucleotide probes to adenylate cyclase-specific mRNA, quantitativeRT-PCR, immunologic detection of a adenylate cyclase polypeptide, ormeasurement of adenylate cyclase activity.

[0240] In any of the embodiments described above, any of thepharmaceutical compositions of the invention can be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy can be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents can actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

[0241] Any of the therapeutic methods described above can be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

Diagnostic Methods

[0242] Human adenylate cyclase also can be used in diagnostic assays fordetecting diseases and abnormalities or susceptibility to diseases andabnormalities related to the presence of mutations in the nucleic acidsequences which encode the enzyme. For example, differences can bedetermined between the cDNA or genomic sequence encoding adenylatecyclase in individuals afflicted with a disease and in normalindividuals. If a mutation is observed in some or all of the afflictedindividuals but not in normal individuals, then the mutation is likelyto be the causative agent of the disease.

[0243] Sequence differences between a reference gene and a gene havingmutations can be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments can be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer can beused with a double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures using radiolabeled nucleotides orby automatic sequencing procedures using fluorescent tags.

[0244] Genetic testing based on DNA sequence differences can be carriedout by detection of alteration in electrophoretic mobility of DNAfragments in gels with or without denaturing agents. Small sequencedeletions and insertions can be visualized, for example, by highresolution gel electrophoresis. DNA fragments of different sequences canbe distinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985).Sequence changes at specific locations can also be revealed by nucleaseprotection assays, such as RNase and S 1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Nat!. Acad. Sci USA 85,4397-4401, 1985). Thus, the detection of a specific DNA sequence can beperformed by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes andSouthern blotting of genomic DNA. In addition to direct methods such asgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

[0245] Altered levels of a adenylate cyclase also can be detected invarious tissues. Assays used to detect levels of the receptorpolypeptides in a body sample, such as blood or a tissue biopsy, derivedfrom a host are well known to those of skill in the art and includeradioimmunoassays, competitive binding assays, Western blot analysis,and ELISA assays.

[0246] All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLE 1

[0247] Detection of Adenylate Cyclase Activity

[0248] The polynucleotide of SEQ ID NO: 4 is inserted into theexpression vector pCEV4 and the expression vector pCEV4-adenylatecyclase polypeptide obtained is transfected into human embryonic kidney293 cells. From these cells extracts are obtained and the adenylatecyclase activity is meassured in the following assay: A volume of 1.0microliter of either H₂O, NaF, guanylyl-5′-imidodiphosphate (GppNHp),isoproterenol, or isoproterenol+GppNHp are added to each of fivereaction tubes and maintained at 0° C. Next, 25 microliter of reactionmixture A (Tris Acetate 100 mM, pH 7.4; KCl 20 mM; MgCl₂ 10.0 mM;phosphoenolpyruvate 20 mM; ATP 2.0 mM; GTP 0.02 mM; dithiothreitol 2.0mM; bovine serum albumin 0.04%; cAMP 0.66 mM; pyruvate kinase 1.0 mg/mland alpha 32 P-ATP, 3000 Ci/mmole) are added to each reaction tube.Finally 25 microliter of the cell extract are added to each tube and thereaction is initiated by placing the tubes in a water bath at 37° C.After 30 minutes, the reaction is terminated by the addition of 300mircroliter of a stopping solution. The assay tubes are heated at 95° C.for 5 minutes. 32 p-cAMP is isolated using Dowex-alumina chromatography.It is shown that the polypeptide of SEQ ID NO: 6 has a adenylate cyclaseactivity.

EXAMPLE 2

[0249] Expression of Recombinant Human Adeynylate Cyclase

[0250] The Pichia pastoris expression vector pPICZB (Invitrogen, SanDiego, Calif.) is used to produce large quantities of recombinant humanadenylate cyclase polypeptides in yeast. The adenylate cyclase-encodingDNA sequence is derived from SEQ ID NO:4 or 5. Before insertion intovector pPICZB, the DNA sequence is modified by well known methods insuch a way that it contains at its 5′-end an initiation codon and at its3′-end an enterokinase cleavage site, a His6 reporter tag and atermination codon.

[0251] Moreover, at both termini recognition sequences for restrictionendonucleases are added and after digestion of the multiple cloning siteof pPICZ B with the corresponding restriction enzymes the modified DNAsequence is ligated into pPICZB. This expression vector is designed forinducible expression in Pichia pastoris, driven by a yeast promoter. Theresulting pPICZ/md-His6 vector is used to transform the yeast.

[0252] The yeast is cultivated under usual conditions in 5 liter shakeflasks and the recombinantly produced protein isolated from the cultureby affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.The bound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humanadenylate cyclase polypeptide is obtained.

EXAMPLE 3

[0253] Identification of Test Compounds that Bind to Adenylate CyclasePolypeptides

[0254] Purified adenylate cyclase polypeptides comprising aglutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Human adenylate cyclase polypeptidescomprise the amino acid sequence shown in SEQ ID NO:6. The testcompounds comprise a fluorescent tag. The samples are incubated for 5minutes to one hour. Control samples are incubated in the absence of atest compound.

[0255] The buffer solution containing the test compounds is washed fromthe wells. Binding of a test compound to a adenylate cyclase polypeptideis detected by fluorescence measurements of the contents of the wells. Atest compound which increases the fluorescence in a well by at least 15%relative to fluorescence of a well in which a test compound is notincubated is identified as a compound which binds to a adenylate cyclasepolypeptide.

EXAMPLE 4

[0256] Identification of a Test Compound which Decreases AdenylateCyclase Gene Expression

[0257] A test compound is administered to a culture of human cellstransfected with a adenylate cyclase expression construct and incubatedat 37° C. for 10 to 45 minutes. A culture of the same type of cellswhich have not been transfected is incubated for the same time withoutthe test compound to provide a negative control.

[0258] RNA is isolated from the two cultures as described in Chirgwin etal., Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20to 30 μg total RNA and hybridized with a ³²P-labeled adenylatecyclase-specific probe at 65° C. in Express-hyb (CLONTECH). The probecomprises at least 11 contiguous nucleotides selected from thecomplement of SEQ D NO:4 or 5. A test compound which decreases theadenylate cyclase-specific signal relative to the signal obtained in theabsence of the test compound is identified as an inhibitor of adenylatecyclase gene expression.

EXAMPLE 5

[0259] Identification of a Test Compound Which Decreases AdenylateCyclase Activity

[0260] A test compound is administered to a culture of human cellstransfected with a adenylate cyclase expression construct and incubatedat 37° C. for 10 to 45 minutes. A culture of the same type of cellswhich have not been transfected is incubated for the same time withoutthe test compound to provide a negative control. Adenylate cyclaseactivity is measured using the method of U.S. Pat. No. 5,795,756.

[0261] A test compound which decreases the enzyme activity of theadenylate cyclase relative to the enzyme activity in the absence of thetest compound is identified as an inhibitor of adenylate cyclaseactivity.

EXAMPLE 6

[0262] Tissue-specific Expression of Adenylate Cyclase

[0263] The qualitative expression pattern of adenylate cyclase invarious tissues is determined by Reverse Transcription-Polymerase ChainReaction (RT-PCR).

[0264] To demonstrate that adenylate cyclase is involved in the diseaseprocess of diabetes, the following whole body panel is screened to showpredominant or relatively high expression: subcutaneous and mesentericadipose tissue, adrenal gland, bone marrow, brain, colon, fetal brain,heart, hypothalamus, kidney, liver, lung, mammary gland, pancreas,placenta, prostate, salivary gland, skeletal muscle, small intestine,spleen, stomach, testis, thymus, thyroid, trachea, and uterus. Humanislet cells and an islet cell library also are tested. As a final step,the expression of adenylate cyclase in cells derived from normalindividuals with the expression of cells derived from diabeticindividuals is compared.

[0265] To demonstrate that adenylate cyclase is involved in the diseaseprocess of COPD, the initial expression panel consists of RNA samplesfrom respiratory tissues and inflammatory cells relevant to COPD: lung(adult and fetal), trachea, freshly isolated alveolar type II cells,cultured human bronchial epithelial cells, cultured small airwayepithelial cells, cultured bronchial sooth muscle cells, cultured H441cells (Clara-like), freshly isolated neutrophils and monocytes, andcultured monocytes (macrophage-like). Body map profiling also is carriedout, using total RNA panels purchased from Clontech. The tissues areadrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung,mammary gland, pancreas, prostate, salivary gland, skeletal muscle,small intestine, spleen, stomach, testis, thymus, trachea, thyroid, anduterus. Quantitative expression profiling. Quantitative expressionprofiling is performed by the form of quantitative PCR analysis called“kinetic analysis” firstly described in Higuchi et al., BioTechnologv10, 413-17, 1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993.The principle is that at any given cycle within the exponential phase ofPCR, the amount of product is proportional to the initial number oftemplate copies.

[0266] If the amplification is performed in the presence of aninternally quenched fluorescent oligonucleotide (TaqMan probe)complementary to the target sequence, the probe is cleaved by the 5′-3′endonuclease activity of Taq DNA polymerase and a fluorescent dyereleased in the medium (Holland et al., Proc. Natl. Acad. Sci. U.S.A.88, 7276-80, 1991). Because the fluorescence emission will increase indirect proportion to the amount of the specific amplified product, theexponential growth phase of PCR product can be detected and used todetermine the initial template concentration (Heid et al., Genome Res.6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).

[0267] The amplification of an endogenous control can be performed tostandardize the amount of sample RNA added to a reaction. In this kindof experiment, the control of choice is the 18S ribosomal RNA. Becausereporter dyes with differing emission spectra are available, the targetand the endogenous control can be independently quantified in the sametube if probes labeled with different dyes are used.

[0268] All “real time PCR” measurements of fluorescence are made in theABI Prism 7700.

[0269] RNA extraction and cDNA preparation. Total RNA from the tissueslisted above are used for expression quantification. RNAs labeled “fromautopsy” were extracted from autoptic tissues with the TRIzol reagent(Life Technologies, MD) according to the manufacturer's protocol. Fiftyμg of each RNA were treated with DNase I for 1 hour at 37° C. in thefollowing reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics,Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mMTris-HCl pH 7.9; 10 mM MgCl₂; 50 mM NaCl; and 1 mM DTT.

[0270] After incubation, RNA is extracted once with 1 volume ofphenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform,and precipitated with 1/10 volume of 3 M NaAcetate, pH 5.2, and 2volumes of ethanol.

[0271] Fifty μg of each RNA from the autoptic tissues are DNase treatedwith the DNA-free kit purchased from Ambion (Ambion, Tex.). Afterresuspension and spectrophotometric quantification, each sample isreverse transcribed with the TaqMan Reverse Transcription Reagents (PEApplied Biosystems, CA) according to the manufacturer's protocol. Thefinal concentration of RNA in the reaction mix is 200 ng/μL. Reversetranscription is carried out with 2.5 μM of random hexamer primers.

[0272] TaqMan quantitative analysis. Specific primers and probe aredesigned according to the recommendations of PE Applied Biosystems; theprobe can be labeled at the 5′ end FAM (6-carboxy-fluorescein) and atthe 3′ end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantificationexperiments are performed on 10 ng of reverse transcribed RNA from eachsample. Each determination is done in triplicate.

[0273] Total cDNA content is normalized with the simultaneousquantification (multiplex PCR) of the 18S ribosomal RNA using thePre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE AppliedBiosystems, CA).

[0274] The assay reaction mix is as follows: 1×final TaqMan UniversalPCR Master Mix (from 2× stock) (PE Applied Biosystems, CA); 1×PDARcontrol-18S RNA (from 20× stock); 300 nM forward primer; 900 nM reverseprimer, 200 nM probe; 10 ng cDNA; and water to 25 μl.

[0275] Each of the following steps are carried out once: pre PCR, 2minutes at 50° C., and 10 minutes at 95° C. The following steps arecarried out 40 times: denaturation, 15 seconds at 95° C.,annealing/extension, 1 minute at 60° C.

[0276] The experiment is performed on an ABI Prism 7700 SequenceDetector (PE Applied Biosystems, CA). At the end of the run,fluorescence data acquired during PCR are processed as described in theABI Prism 7700 user's manual in order to achieve better backgroundsubtraction as well as signal linearity with the starting targetquantity.

[0277] Expression of human adenylate cyclase in liver, skeletal muscle,hypothalamus, islet cells, and adipose tissue is shown in FIG. 51.

EXAMPLE 7

[0278] Diabetes: In vivo Testing of Compounds/Target Validation

[0279] 1. Glucose Production:

[0280] Over-production of glucose by the liver, due to an enhanced rateof gluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

[0281] 2. Insulin Sensitivity:

[0282] Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

[0283] 3. Insulin Secretion:

[0284] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, compounds are administered by different routes (p.o.,i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At theappropriate time an intravenous glucose load (0.4 g/kg) is given, bloodis collected one minute later. Plasma insulin levels are determined.Compounds that enhance insulin secretion will increase plasma insulinlevels compared to animals given only glucose. When measuring glucosedisappearance, animals are bled at the appropriate time after compoundadministration, then given either an oral or intraperitoneal glucoseload (1 g/kg), bled again after 15, 30, 60 and 90 minutes and plasmaglucose levels determined. Compounds that increase insulin levels willdecrease glucose levels and the area-under-the glucose curve whencompared to the vehicle-treated group given only glucose.

[0285] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, test compounds which regulate adenylate cyclase areadministered by different routes (p.o., i.p., s.c., or i.v.) toovernight fasted normal rats or mice. At the appropriate time anintravenous glucose load (0.4 g/kg) is given, blood is collected oneminute later. Plasma insulin levels are determined. Test compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60, and 90 minutes and plasma glucose levelsdetermined. Test compounds that increase insulin levels will decreaseglucose levels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

[0286] 4. Glucose Production:

[0287] Over-production of glucose by the liver, due to an enhanced rateof gluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

[0288] 5. Insulin Sensitivity:

[0289] Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

[0290] 6. Insulin Secretion:

[0291] Compounds that enhance insulin secretion from the pancreas willincrease plasma insulin levels and improve the disappearance of plasmaglucose following the administration of a glucose load. When measuringinsulin levels, compounds are administered by different routes (p.o.,i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At theappropriate time an intravenous glucose load (0.4 g/kg) is given, bloodis collected one minute later. Plasma insulin levels are determined.Compounds that enhance insulin secretion will increase plasma insulinlevels compared to animals given only glucose. When measuring glucosedisappearance, animals are bled at the appropriate time after compoundadministration, then given either an oral or intraperitoneal glucoseload (1 g/kg), bled again after 15, 30, 60 and 90 minutes and plasmaglucose levels determined. Compounds that increase insulin levels willdecrease glucose levels and the area-under-the glucose curve whencompared to the vehicle-treated group given only glucose.

EXAMPLE 8

[0292] In Vivo Testing of Compounds/Target Validation

[0293] 1. Pain:

[0294] Acute Pain Acute pain is measured on a hot plate mainly in rats.Two variants of hot plate testing are used: In the classical variantanimals are put on a hot surface (52 to 56 μC) and the latency time ismeasured until the animals show nocifensive behavior, such as steppingor foot licking. The other variant is an increasing temperature hotplate where the experimental animals are put on a surface of neutraltemperature. Subsequently this surface is slowly but constantly heateduntil the animals begin to lick a hind paw. The temperature which isreached when hind paw licking begins is a measure for pain threshold.

[0295] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., p.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to pain testing.

Persistent Pain

[0296] Persistent pain is measured with the formalin or capsaicin test,mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicinis injected into one hind paw of the experimental animal. After formalinor capsaicin application the animals show nocifensive reactions likeflinching, licking and biting of the affected paw. The number ofnocifensive reactions within a time frame of up to 90 minutes is ameasure for intensity of pain.

[0297] Compounds are tested against a vehicle treated control group.Substance application is performed at different time points viadifferent application routes (i.v., i.p., P.o., i.t., i.c.v., s.c.,intradermal, transdermal) prior to formalin or capsaicin administration.

Neuropathic Pain

[0298] Neuropathic pain is induced by different variants of unilateralsciatic nerve injury mainly in rats. The operation is performed underanesthesia The first variant of sciatic nerve injury is produced byplacing loosely constrictive ligatures around the common sciatic nerve.The second variant is the tight ligation of about the half of thediameter of the common sciatic nerve. In the next variant, a group ofmodels is used in which tight ligations or transections are made ofeither the L5 and L6 spinal nerves, or the L % spinal nerve only. Thefourth variant involves an axotomy of two of the three terminal branchesof the sciatic nerve (tibial and common peroneal nerves) leaving theremaining sural nerve intact whereas the last variant comprises theaxotomy of only the tibial branch leaving the sural and common nervesuninjured. Control animals are treated with a sham operation.

[0299] Postoperatively, the nerve injured animals develop a chronicmechanical allodynia, cold allodynioa, as well as a thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA; Electronic von Frey System,Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is measured bymeans of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy), or by means of a cold plate of 5 to 10 C where the nocifensivereactions of the affected hind paw are counted as a measure of painintensity. A further test for cold induced pain is the counting ofnocifensive reactions, or duration of nocifensive responses afterplantar administration of acetone to the affected hind limb. Chronicpain in general is assessed by registering the circadanian rhythms inactivity (Surjo and Arndt, Universität zu Köln, Cologne, Germany), andby scoring differences in gait (foot print patterns; FOOTPRINTS program,Klapdor et al., 1997. A low cost method to analyze footprint patterns.J. Neurosci. Methods 75, 49-54).

[0300] Compounds are tested against sham operated and vehicle treatedcontrol groups. Substance application is performed at different timepoints via different application routes (i.v., i.p., p.o., i.t., i.c.v.,S.c., intradermal, transdermal) prior to pain testing.

Inflammatory Pain

[0301] Inflammatory pain is induced mainly in rats by injection of 0.75mg carrageenan or complete Freund's adjuvant into one hind paw. Theanimals develop an edema with mechanical allodynia as well as thermalhyperalgesia. Mechanical allodynia is measured by means of a pressuretransducer (electronic von Frey Anesthesiometer, IITC Inc.-Life ScienceInstruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measuredby means of a radiant heat source (Plantar Test, Ugo Basile, Comerio,Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).For edema measurement two methods are being used. In the first method,the animals are sacrificed and the affected hindpaws sectioned andweighed. The second method comprises differences in paw volume bymeasuring water displacement in a plethysmometer (Ugo Basile, Comerio,Italy).

[0302] Compounds are tested against uninflamed as well as vehicletreated control groups. Substance application is performed at differenttime points via different application routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

Diabetic Neuropathic Pain

[0303] Rats treated with a single intraperitoneal injection of 50 to 80mg/kg streptozotocin develop a profound hyperglycemia and mechanicalallodynia within 1 to 3 weeks. Mechanical allodynia is measured by meansof a pressure transducer (electronic von Frey Anesthesiometer, IITCInc.-Life Science Instruments, Woodland Hills, SA, USA).

[0304] Compounds are tested against diabetic and non-diabetic vehicletreated control groups. Substance application is performed at differenttime points via different application routes (i.v., i.p., p.o., i.t.,i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0305] 2. Parkinson's Disease

[0306] 6-Hydroxydopamine (6-OH-DA) Lesion

[0307] Degeneration of the dopaminergic nigrostriatal andstriatopallidal pathways is the central pathological event inParkinson's disease. This disorder has been mimicked experimentally inrats using single/sequential unilateral stereotaxic injections of6-OH-DA into the medium forebrain bundle (MFB).

[0308] Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 gat the beginning of the experiment, are used. The rats are maintained ina temperature- and humidity-controlled environment under a 12 hlight/dark cycle with free access to food and water when not inexperimental sessions. The following in vivo protocols are approved bythe governmental authorities. All efforts are made to minimize animalsuffering, to reduce the number of animals used, and to utilizealternatives to in vivo techniques.

[0309] Animals are administered pargyline on the day of surgery (Sigma,St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of6-OHDA by monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kgi.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals.Thirty minutes later the rats are anesthetized with sodium pentobarbital(50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DAnigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μgof 6-OHDA HBr (Sigma) are injected into the left medial fore-brainbundle at a rate of 1 μl/min (2.4 mrn anterior, 1.49 mm lateral, −2.7 mmventral to Bregma and the skull surface). The needle is left in place anadditional 5 min to allow diffusion to occur.

[0310] Stepping Test

[0311] Forelimb akinesia is assessed three weeks following lesionplacement using a modified stepping test protocol. In brief, the animalsare held by the experimenter with one hand fixing the hindlimbs andslightly raising the hind part above the surface. One paw is touchingthe table, and is then moved slowly sideways (5 s for 1 m), first in theforehand and then in the backhand direction. The number of adjustingsteps is counted for both paws in the backhand and forehand direction ofmovement. The sequence of testing is right paw forehand and backhandadjusting stepping, followed by left paw forehand and backhanddirections. The test is repeated three times on three consecutive days,after an initial training period of three days prior to the firsttesting. Forehand adjusted stepping reveals no consistent differencesbetween lesioned and healthy control animals. Analysis is thereforerestricted to backhand adjusted stepping.

[0312] Balance Test

[0313] Balance adjustments following postural challenge are alsomeasured during the stepping test sessions. The rats are held in thesame position as described in the stepping test and, instead of beingmoved sideways, tilted by the experimenter towards the side of the pawtouching the table. This maneuver results in loss of balance and theability of the rats to regain balance by forelimb movements is scored ona scale ranging from 0 to 3. Score 0 is given for a normal forelimbplacement. When the forelimb movement is delayed but recovery ofpostural balance detected, score 1 is given. Score 2 represents a clear,yet insufficient, forelimb reaction, as evidenced by muscle contraction,but lack of success in recovering balance, and score 3 is given for noreaction of movement. The test is repeated three times a day on eachside for three consecutive days after an initial training period ofthree days prior to the first testing.

[0314] Staircase Test (Paw Reaching)

[0315] A modified version of the staircase test is used for evaluationof paw reaching behavior three weeks following primary and secondarylesion placement. Plexiglass test boxes with a central platform and aremovable staircase on each side are used. The apparatus is designedsuch that only the paw on the same side at each staircase can be used,thus providing a measure of independent forelimb use. For each test theanimals are left in the test boxes for 15 min. The double staircase isfilled with 7×3 chow pellets (Precision food pellets, formula: P,purified rodent diet, size 45 mg; Sandown Scientific) on each side.After each test the number of pellets eaten (successfully retrievedpellets) and the number of pellets taken (touched but dropped) for eachpaw and the success rate (pellets eaten/pellets taken) are countedseparately. After three days of food deprivation (12 g per animal perday) the animals are tested for 11 days. Full analysis is conducted onlyfor the last five days.

[0316] MPTP Treatment

[0317] The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP) causes degeneration of mesencephalic dopaminergic (DAergic)neurons in rodents, non-human primates, and humans and, in so doing,reproduces many of the symptoms of Parkinson's disease. MPTP leads to amarked decrease in the levels of dopamine and its metabolites, and inthe number of dopaminergic terminals in the striatum as well as severeloss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in thesubstantia nigra, pars compacta

[0318] In order to obtain severe and long-lasting lesions, and to reducemortality, animals receive single injections of MPTP, and are thentested for severity of lesion 7-10 days later. Successive MPTPinjections are administered on days 1, 2 and 3. Animals receiveapplication of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily.All injections are intraperitoneal (i.p.) and the MPTP stock solution isfrozen between injections. Animals are decapitated on day 11.

[0319] Immunohistology

[0320] At the completion of behavioral experiments, all animals areanaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). Themice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min,followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brainsare removed and placed in 4% paraformaldehyde for 24 h at 4° C. Fordehydration they are then transferred to a 20% sucrose (Merck) solutionin 0.1 M PBS at 4° C. until they sink. The brains are frozen inmethylbutan at −20° C. for 2 min and stored at −70° C. Using a sledgemicrotome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from thegenu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm)and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored inassorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry. Aseries of sections is processed for free-floating tyrosine hydroxylase(TH) immunohistochemistry. Following three rinses in 0.1 M PBS,endogenous peroxi-dase activity is quenched for 10 min in 0.3% H₂O₂±PBS.After rinsing in PBS, sections are preincubated in 10% normal bovineserum (Sigma) for 5 min as blocking agent and transferred to eitherprimary anti-rat TH rabbit antiserum (dilution 1:2000).

[0321] Following overnight incubation at room temperature, sections forTH immuno-reactivity are rinsed in PBS (2×10 min) and incubated inbiotinylated anti-rabbit immunoglobulin G raised in goat (dilution1:200) (Vector) for 90 min, rinsed repeatedly and transferred toVectastain ABC (Vector) solution for 1 h. 3,.3′-Diaminobenzidinetetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%H₂O₂, serves as chromogen in the subsequent visualization reaction.Sections are mounted on to gelatin-coated slides, left to dry overnight,counter-stained with hematoxylin dehydrated in ascending alcoholconcentrations and cleared in butylacetate. Coverslips are mounted onentellan.

[0322] Rotarod Test

[0323] We use a modification of the procedure described by Rozas andLabandeira-Garcia (1997), with a CR-1 Rotamex system (ColumbusInstruments, Columbus, Ohio) comprising an IBM-compatible personalcomputer, a CIO-24 data acquisition card, a control unit, and afour-lane rotarod unit. The rotarod unit consists of a rotating spindle(diameter 7.3 cm) and individual compartments for each mouse. The systemsoftware allows preprogramming of session protocols with varyingrotational speeds (0-80 rpm). Infrared beams are used to detect when amouse has fallen onto the base grid beneath the rotarod. The system logsthe fall as the end of the experiment for that mouse, and the total timeon the rotarod, as well as the time of the fall and all the set-upparameters, are recorded. The system also allows a weak current to bepassed through the base grid, to aid training.

[0324] 3. Dementia

[0325] The Object Recognition Task

[0326] The object recognition task has been designed to assess theeffects of experimental manipulations on the cognitive performance ofrodents. A rat is placed in an open field, in which two identicalobjects are present. The rats inspects both objects during the firsttrial of the object recognition task. In a second trial, after aretention interval of for example 24 hours, one of the two objects usedin the first trial, the ‘familiar’ object, and a novel object are placedin the open field. The inspection time at each of the objects isregistered. The basic measures in the OR task is the time spent by a ratexploring the two object the second trial. Good retention is reflectedby higher exploration times towards the novel than the ‘familiar’object.

[0327] Administration of the putative cognition enhancer prior to thefirst trial predo-minantly allows assessment of the effects onacquisition, and eventually on consolidation processes. Administrationof the testing compound after the first trial allows to assess theeffects on consolidation processes, whereas administration before thesecond trial allows to measure effects on retrieval processes.

[0328] The Passive Avoidance Task

[0329] The passive avoidance task assesses memory performance in ratsand mice. The inhibitory avoidance apparatus consists of atwo-compartment box with a light compartment and a dark compartment. Thetwo compartments are separated by a guillotine door that can be operatedby the experimenter. A threshold of 2 cm separates the two compartmentswhen the guillotine door is raised. When the door is open, theillumination in the dark compartment is about 2 lux. The light intensityis about 500 lux at the center of the floor of the light compartment.

[0330] Two habituation sessions, one shock session, and a retentionsession are given, separated by inter-session intervals of 24 hours. Inthe habituation sessions and the retention session the rat is allowed toexplore the apparatus for 300 sec. The rat is placed in the lightcompartment, facing the wall opposite to the guillotine door. After anaccommodation period of 15 sec. the guillotine door is opened so thatall parts of the apparatus can be visited freely. Rats normally avoidbrightly lit areas and will enter the dark compartment within a fewseconds.

[0331] In the shock session the guillotine door between the compartmentsis lowered as soon as the rat has entered the dark compartment with itsfour paws, and a scrambled 1 mA footshock is administered for 2 sec. Therat is removed from the apparatus and put back into its home cage. Theprocedure during the retention session is identical to that of thehabituation sessions.

[0332] The step-through latency, that is the first latency of enteringthe dark compartment (in sec.) during the retention session is an indexof the memory performance of the animal; the longer the latency to enterthe dark compartment, the better the retention is. A testing compound ingiven half an hour before the shock session, together with 1 mg*kg⁻¹scopolamine. Scopolamine impairs the memory performance during theretention session 24 hours later. If the test compound increases theenter latency compared with the scopolamine-treated controls, is likelyto possess cognition enhancing potential.

[0333] The Morris Water Escape Task

[0334] The Morris water escape task measures spatial orientationlearning in rodents. It is a test system that has extensively been usedto investigate the effects of putative therapeutic on the cognitivefunctions of rats and mice. The performance of an animal is assessed ina circular water tank with an escape platform that is submerged about 1cm below the surface of the water. The escape platform is not visiblefor an animal swimming in the water tank. Abundant extra-maze cues areprovided by the furniture in the room, including desks, computerequipment, a second water tank, the presence of the experimenter, and bya radio on a shelf that is playing softly.

[0335] The animals receive four trials during five daily acquisitionsessions. A trial is started by placing an animal into the pool, facingthe wall of the tank. Each of four starting positions in the quadrantsnorth, east, south, and west is used once in a series of four trials;their order is randomized. The escape platform is always in the sameposition. A trial is terminated as soon as the animal had climbs ontothe escape platform or when 90 seconds have elapsed, whichever eventoccurs first. The animal is allowed to stay on the platform for 30seconds. Then it is taken from the platform and the next trial isstarted. If an animal did not find the platform within 90 seconds it isput on the platform by the experimenter and is allowed to stay there for30 seconds. After the fourth trial of the fifth daily session, anadditional trial is given as a probe trial: the platform is removed, andthe time the animal spends in the four quadrants is measured for 30 or60 seconds. In the probe trial, all animals start from the same startposition, opposite to the quadrant where the escape platform had beenpositioned during acquisition.

[0336] Four different measures are taken to evaluate the performance ofan animal during acquisition training: escape latency, traveleddistance, distance to platform, and swimming speed. The followingmeasures are evaluated for the probe trial: time (s) in quadrants andtraveled distance (cm) in the four quadrants. The probe trial providesadditional information about how well an animal learned the position ofthe escape platform. If an animal spends more time and swims a longerdistance in the quadrant where the platform had been positioned duringthe acquisition sessions than in any other quadrant, one concludes thatthe platform position has been learned well.

[0337] In order to assess the effects of putative cognition enhancingcompounds, rats or mice with specific brain lesions which impaircognitive functions, or animals treated with compounds such asscopolamine or MK-801, which interfere with normal learning, or agedanimals which suffer from cognitive deficits, are used.

[0338] The T-maze Spontaneous Alternation Task

[0339] The T-maze spontaneous alternation task (TeMCAT) assesses thespatial memory performance in mice. The start arm and the two goal armsof the T-maze are provided with guillotine doors which can be operatedmanually by the experimenter. A mouse is put into the start arm at thebeginning of training. The guillotine door is closed. In the firsttrial, the ‘forced trial’, either the left or right goal arm is blockedby lowering the guillotine door. After the mouse has been released fromthe start arm, it will negotiate the maze, eventually enter the opengoal arm, and return to the start position, where it will be confinedfor 5 seconds, by lowering the guillotine door. Then, the animal canchoose freely between the left and right goal arm (all guillotine-doorsopened) during 14 ‘free choice’ trials. As soon a the mouse has enteredone goal arm the other one is closed. The mouse eventually returns tothe start arm and is free to visit whichever go alarm it wants afterhaving been confined to the start arm for 5 seconds. After completion of14 free choice trials in one session, the animal is removed from themaze. During training, the animal is never handled.

[0340] The percent alternations out of 14 trials is calculated. Thispercentage and the total time needed to complete the first forced trialand the subsequent 14 free choice trials (in s) is analyzed. Cognitivedeficits are usually induced by an injection of scopolamine, 30 minbefore the start of the training session. Scopolamine reduced theper-cent alternations to chance level, or below. A cognition enhancer,which is always administered before the training session, will at leastpartially, antagonize the scopolamine-induced reduction in thespontaneous alternation rate.

1 7 1 2664 DNA Homo sapiens 1 ggagcctacc ataagcacct catggaactcgctcttcagc aaacatatca ggacacctgt 60 aattgcatca agtcgcggat caagttggaatttgaaaaac gtcaacagga gcggcttctg 120 ctctccctgc tgccggccca catcgccatggagatgaaag cggagatcat ccagaggctg 180 cagggcccca aggcgggcca gatggagaacacaaataact tccacaacct gtatgtgaag 240 cggcatacaa acgtgagcat cttatacgctgacatcgttg gctttacccg gctggcaagt 300 gactgctccc cgggagaact agtccacatgctgaatgagc tctttggaaa gtttgatcaa 360 attgcaaagg agaatgaatg catgagaattaaaattttag gagactgcta ctactgtgta 420 tctggactcc ctatatctct ccctaaccatgccaagaact gtgtgaaaat ggggctggac 480 atgtgtgaag ccataaagaa agtgagggatgctactggag ttgatatcaa catgcgcgtg 540 ggcgtgcatt ctgggaatgt cctgtgtggcgtgattggtc tgcagaagtg gcaatatgat 600 gtgtggtcac atgatgtgac cttggccaaccacatggaag ctggaggggt ccctggacgt 660 gttcacattt cttctgtcac cctggagcacttgaatggcg cttataaagt ggaggaggga 720 gatggtgaca ttagggaccc atatttaaaacagcacctgg tgaaaaccta ctttgtgatc 780 aaccccaagg gagaacgacg gagcccccagcatctcttca gacctcgcca cacccttgat 840 ggagccaaaa tgagggcctc ggtccgcatgacccggtact tggagtcctg gggggcagcc 900 aagccctttg cacacctaca tcacagggacagcatgacca cagagaacgg caagatcagc 960 accacggatg tacccatggg tcagcataattttcaaaatc gcaccttaag aaccaagtca 1020 caaaagaaga gatttgaaga agaattgaatgaaaggatga ttcaagcaat tgatgggatt 1080 aatgcacaga agcaatggct caagtctgaagacattcaga gaatctcact gcttttctat 1140 aacaaagtac tagaaaaaga gtaccgggccacggcactgc cagcgttcaa gtattatgtg 1200 acttgtgcct gtctcatatt cttctgcatcttcattgtgc agattctcgt gctgccaaaa 1260 acgtctgtcc tgggcatctc ctttggggctgcgtttctct tgctggcctt catcctcttc 1320 gtctgctttg ctggacagct tctgcaatgcagcaaaaaag cctctcccct gctcatgtgg 1380 cttttgaagt cctcgggcat cattgccaaccgcccctggc cacggatctc tctcacgatc 1440 atcaccacag ccatcatatt aatgatggccgtgttcaaca tgtttttcct gagtgactca 1500 gaggaaacaa tccctccaac tgccaacacaacaaacacaa gcttttcagc ctcaaataat 1560 caggtggcga ttctgcgtgc gcagaatttatttttcctcc cgtactttat ctacagctgc 1620 attctgggac tgatatcctg ttccgtgttcctgcgggtaa actatgagct gaagatgttg 1680 atcatgatgg tggccttggt gggctacaacaccatcctac tccacaccca cgcccacgtc 1740 ctgggcgact acagccaggt cttatttgagagaccaggca tttggaaaga cctgaagacc 1800 atgggctctg tgtctctctc tatattcttcatcacactgc ttgttctggg tagacagaat 1860 gaatattact gtaggttaga cttcttatggaagaacaaat tcaaaaaaga gcgggaggag 1920 atagagacca tggagaacct gaaccgcgtgctgctggaga acgtgcttcc cgcgcacgtg 1980 gctgagcact tcctggccag gagcctgaagaatgaggagc tataccacca gtcctatgac 2040 tgcgtctgtg tcatgtttgc ctccattccggatttcaaag aattttatac agaatccgac 2100 gtgaacaagg agggcttgga atgccttcggctcctgaacg agatcatcgc tgactttgat 2160 gatcttcttt ccaagccaaa attcagtggagttgaaaaga ttaagaccat tggcagcaca 2220 tacatggcag caacaggtct gagcgctgtgcccagccagg agcactccca ggagcccgag 2280 cggcagtaca tgcacattgg caccatggtggagtttgctt ttgccctggt agggaagctg 2340 gatgccatca acaagcactc cttcaacgacttcaaattgc gagtgggtat taaccatgga 2400 cctgtgatag ctggtgtgat tggagctcagaagccacaat atgatatctg gggcaacact 2460 gtcaatgtgg ccagtaggat ggacagcaccggagtcctgg acaaaataca ggttaccgag 2520 gagacgagcc tcgtcctgca gaccctcggatacacgtgca cctgtcgagg aataatcaac 2580 gtgaaaggaa agggggacct gaagacgtactttgtaaaca cagaaatgtc aaggtccctt 2640 tcccagagca acgtggcatc ctga 2664 25873 DNA Homo sapiens CDS (1)..(2664) 2 gga gcc tac cat aag cac ctc atggaa ctc gct ctt cag caa aca tat 48 Gly Ala Tyr His Lys His Leu Met GluLeu Ala Leu Gln Gln Thr Tyr 1 5 10 15 cag gac acc tgt aat tgc atc aagtcg cgg atc aag ttg gaa ttt gaa 96 Gln Asp Thr Cys Asn Cys Ile Lys SerArg Ile Lys Leu Glu Phe Glu 20 25 30 aaa cgt caa cag gag cgg ctt ctg ctctcc ctg ctg ccg gcc cac atc 144 Lys Arg Gln Gln Glu Arg Leu Leu Leu SerLeu Leu Pro Ala His Ile 35 40 45 gcc atg gag atg aaa gcg gag atc atc cagagg ctg cag ggc ccc aag 192 Ala Met Glu Met Lys Ala Glu Ile Ile Gln ArgLeu Gln Gly Pro Lys 50 55 60 gcg ggc cag atg gag aac aca aat aac ttc cacaac ctg tat gtg aag 240 Ala Gly Gln Met Glu Asn Thr Asn Asn Phe His AsnLeu Tyr Val Lys 65 70 75 80 cgg cat aca aac gtg agc atc tta tac gct gacatc gtt ggc ttt acc 288 Arg His Thr Asn Val Ser Ile Leu Tyr Ala Asp IleVal Gly Phe Thr 85 90 95 cgg ctg gca agt gac tgc tcc ccg gga gaa cta gtccac atg ctg aat 336 Arg Leu Ala Ser Asp Cys Ser Pro Gly Glu Leu Val HisMet Leu Asn 100 105 110 gag ctc ttt gga aag ttt gat caa att gca aag gagaat gaa tgc atg 384 Glu Leu Phe Gly Lys Phe Asp Gln Ile Ala Lys Glu AsnGlu Cys Met 115 120 125 aga att aaa att tta gga gac tgc tac tac tgt gtatct gga ctc cct 432 Arg Ile Lys Ile Leu Gly Asp Cys Tyr Tyr Cys Val SerGly Leu Pro 130 135 140 ata tct ctc cct aac cat gcc aag aac tgt gtg aaaatg ggg ctg gac 480 Ile Ser Leu Pro Asn His Ala Lys Asn Cys Val Lys MetGly Leu Asp 145 150 155 160 atg tgt gaa gcc ata aag aaa gtg agg gat gctact gga gtt gat atc 528 Met Cys Glu Ala Ile Lys Lys Val Arg Asp Ala ThrGly Val Asp Ile 165 170 175 aac atg cgc gtg ggc gtg cat tct ggg aat gtcctg tgt ggc gtg att 576 Asn Met Arg Val Gly Val His Ser Gly Asn Val LeuCys Gly Val Ile 180 185 190 ggt ctg cag aag tgg caa tat gat gtg tgg tcacat gat gtg acc ttg 624 Gly Leu Gln Lys Trp Gln Tyr Asp Val Trp Ser HisAsp Val Thr Leu 195 200 205 gcc aac cac atg gaa gct gga ggg gtc cct ggacgt gtt cac att tct 672 Ala Asn His Met Glu Ala Gly Gly Val Pro Gly ArgVal His Ile Ser 210 215 220 tct gtc acc ctg gag cac ttg aat ggc gct tataaa gtg gag gag gga 720 Ser Val Thr Leu Glu His Leu Asn Gly Ala Tyr LysVal Glu Glu Gly 225 230 235 240 gat ggt gac att agg gac cca tat tta aaacag cac ctg gtg aaa acc 768 Asp Gly Asp Ile Arg Asp Pro Tyr Leu Lys GlnHis Leu Val Lys Thr 245 250 255 tac ttt gtg atc aac ccc aag gga gaa cgacgg agc ccc cag cat ctc 816 Tyr Phe Val Ile Asn Pro Lys Gly Glu Arg ArgSer Pro Gln His Leu 260 265 270 ttc aga cct cgc cac acc ctt gat gga gccaaa atg agg gcc tcg gtc 864 Phe Arg Pro Arg His Thr Leu Asp Gly Ala LysMet Arg Ala Ser Val 275 280 285 cgc atg acc cgg tac ttg gag tcc tgg ggggca gcc aag ccc ttt gca 912 Arg Met Thr Arg Tyr Leu Glu Ser Trp Gly AlaAla Lys Pro Phe Ala 290 295 300 cac cta cat cac agg gac agc atg acc acagag aac ggc aag atc agc 960 His Leu His His Arg Asp Ser Met Thr Thr GluAsn Gly Lys Ile Ser 305 310 315 320 acc acg gat gta ccc atg ggt cag cataat ttt caa aat cgc acc tta 1008 Thr Thr Asp Val Pro Met Gly Gln His AsnPhe Gln Asn Arg Thr Leu 325 330 335 aga acc aag tca caa aag aag aga tttgaa gaa gaa ttg aat gaa agg 1056 Arg Thr Lys Ser Gln Lys Lys Arg Phe GluGlu Glu Leu Asn Glu Arg 340 345 350 atg att caa gca att gat ggg att aatgca cag aag caa tgg ctc aag 1104 Met Ile Gln Ala Ile Asp Gly Ile Asn AlaGln Lys Gln Trp Leu Lys 355 360 365 tct gaa gac att cag aga atc tca ctgctt ttc tat aac aaa gta cta 1152 Ser Glu Asp Ile Gln Arg Ile Ser Leu LeuPhe Tyr Asn Lys Val Leu 370 375 380 gaa aaa gag tac cgg gcc acg gca ctgcca gcg ttc aag tat tat gtg 1200 Glu Lys Glu Tyr Arg Ala Thr Ala Leu ProAla Phe Lys Tyr Tyr Val 385 390 395 400 act tgt gcc tgt ctc ata ttc ttctgc atc ttc att gtg cag att ctc 1248 Thr Cys Ala Cys Leu Ile Phe Phe CysIle Phe Ile Val Gln Ile Leu 405 410 415 gtg ctg cca aaa acg tct gtc ctgggc atc tcc ttt ggg gct gcg ttt 1296 Val Leu Pro Lys Thr Ser Val Leu GlyIle Ser Phe Gly Ala Ala Phe 420 425 430 ctc ttg ctg gcc ttc atc ctc ttcgtc tgc ttt gct gga cag ctt ctg 1344 Leu Leu Leu Ala Phe Ile Leu Phe ValCys Phe Ala Gly Gln Leu Leu 435 440 445 caa tgc agc aaa aaa gcc tct cccctg ctc atg tgg ctt ttg aag tcc 1392 Gln Cys Ser Lys Lys Ala Ser Pro LeuLeu Met Trp Leu Leu Lys Ser 450 455 460 tcg ggc atc att gcc aac cgc ccctgg cca cgg atc tct ctc acg atc 1440 Ser Gly Ile Ile Ala Asn Arg Pro TrpPro Arg Ile Ser Leu Thr Ile 465 470 475 480 atc acc aca gcc atc ata ttaatg atg gcc gtg ttc aac atg ttt ttc 1488 Ile Thr Thr Ala Ile Ile Leu MetMet Ala Val Phe Asn Met Phe Phe 485 490 495 ctg agt gac tca gag gaa acaatc cct cca act gcc aac aca aca aac 1536 Leu Ser Asp Ser Glu Glu Thr IlePro Pro Thr Ala Asn Thr Thr Asn 500 505 510 aca agc ttt tca gcc tca aataat cag gtg gcg att ctg cgt gcg cag 1584 Thr Ser Phe Ser Ala Ser Asn AsnGln Val Ala Ile Leu Arg Ala Gln 515 520 525 aat tta ttt ttc ctc ccg tacttt atc tac agc tgc att ctg gga ctg 1632 Asn Leu Phe Phe Leu Pro Tyr PheIle Tyr Ser Cys Ile Leu Gly Leu 530 535 540 ata tcc tgt tcc gtg ttc ctgcgg gta aac tat gag ctg aag atg ttg 1680 Ile Ser Cys Ser Val Phe Leu ArgVal Asn Tyr Glu Leu Lys Met Leu 545 550 555 560 atc atg atg gtg gcc ttggtg ggc tac aac acc atc cta ctc cac acc 1728 Ile Met Met Val Ala Leu ValGly Tyr Asn Thr Ile Leu Leu His Thr 565 570 575 cac gcc cac gtc ctg ggcgac tac agc cag gtc tta ttt gag aga cca 1776 His Ala His Val Leu Gly AspTyr Ser Gln Val Leu Phe Glu Arg Pro 580 585 590 ggc att tgg aaa gac ctgaag acc atg ggc tct gtg tct ctc tct ata 1824 Gly Ile Trp Lys Asp Leu LysThr Met Gly Ser Val Ser Leu Ser Ile 595 600 605 ttc ttc atc aca ctg cttgtt ctg ggt aga cag aat gaa tat tac tgt 1872 Phe Phe Ile Thr Leu Leu ValLeu Gly Arg Gln Asn Glu Tyr Tyr Cys 610 615 620 agg tta gac ttc tta tggaag aac aaa ttc aaa aaa gag cgg gag gag 1920 Arg Leu Asp Phe Leu Trp LysAsn Lys Phe Lys Lys Glu Arg Glu Glu 625 630 635 640 ata gag acc atg gagaac ctg aac cgc gtg ctg ctg gag aac gtg ctt 1968 Ile Glu Thr Met Glu AsnLeu Asn Arg Val Leu Leu Glu Asn Val Leu 645 650 655 ccc gcg cac gtg gctgag cac ttc ctg gcc agg agc ctg aag aat gag 2016 Pro Ala His Val Ala GluHis Phe Leu Ala Arg Ser Leu Lys Asn Glu 660 665 670 gag cta tac cac cagtcc tat gac tgc gtc tgt gtc atg ttt gcc tcc 2064 Glu Leu Tyr His Gln SerTyr Asp Cys Val Cys Val Met Phe Ala Ser 675 680 685 att ccg gat ttc aaagaa ttt tat aca gaa tcc gac gtg aac aag gag 2112 Ile Pro Asp Phe Lys GluPhe Tyr Thr Glu Ser Asp Val Asn Lys Glu 690 695 700 ggc ttg gaa tgc cttcgg ctc ctg aac gag atc atc gct gac ttt gat 2160 Gly Leu Glu Cys Leu ArgLeu Leu Asn Glu Ile Ile Ala Asp Phe Asp 705 710 715 720 gat ctt ctt tccaag cca aaa ttc agt gga gtt gaa aag att aag acc 2208 Asp Leu Leu Ser LysPro Lys Phe Ser Gly Val Glu Lys Ile Lys Thr 725 730 735 att ggc agc acatac atg gca gca aca ggt ctg agc gct gtg ccc agc 2256 Ile Gly Ser Thr TyrMet Ala Ala Thr Gly Leu Ser Ala Val Pro Ser 740 745 750 cag gag cac tcccag gag ccc gag cgg cag tac atg cac att ggc acc 2304 Gln Glu His Ser GlnGlu Pro Glu Arg Gln Tyr Met His Ile Gly Thr 755 760 765 atg gtg gag tttgct ttt gcc ctg gta ggg aag ctg gat gcc atc aac 2352 Met Val Glu Phe AlaPhe Ala Leu Val Gly Lys Leu Asp Ala Ile Asn 770 775 780 aag cac tcc ttcaac gac ttc aaa ttg cga gtg ggt att aac cat gga 2400 Lys His Ser Phe AsnAsp Phe Lys Leu Arg Val Gly Ile Asn His Gly 785 790 795 800 cct gtg atagct ggt gtg att gga gct cag aag cca caa tat gat atc 2448 Pro Val Ile AlaGly Val Ile Gly Ala Gln Lys Pro Gln Tyr Asp Ile 805 810 815 tgg ggc aacact gtc aat gtg gcc agt agg atg gac agc acc gga gtc 2496 Trp Gly Asn ThrVal Asn Val Ala Ser Arg Met Asp Ser Thr Gly Val 820 825 830 ctg gac aaaata cag gtt acc gag gag acg agc ctc gtc ctg cag acc 2544 Leu Asp Lys IleGln Val Thr Glu Glu Thr Ser Leu Val Leu Gln Thr 835 840 845 ctc gga tacacg tgc acc tgt cga gga ata atc aac gtg aaa gga aag 2592 Leu Gly Tyr ThrCys Thr Cys Arg Gly Ile Ile Asn Val Lys Gly Lys 850 855 860 ggg gac ctgaag acg tac ttt gta aac aca gaa atg tca agg tcc ctt 2640 Gly Asp Leu LysThr Tyr Phe Val Asn Thr Glu Met Ser Arg Ser Leu 865 870 875 880 tcc cagagc aac gtg gca tcc tga agagtcacct tcattttggc aagaagactg 2694 Ser GlnSer Asn Val Ala Ser 885 tattttcagg aaggtatcac acactttctg actgcaacttctgtcccttg tttttgatgt 2754 gcgtgctgtc tgtcctatgg agcctctgca gactcgttctcgtgacccag tggcataccg 2814 tttggtgtct gatgtgtgcc cagatcgttc tgccacttgcactgtgcttg ctcctaagca 2874 aaagggaaaa ggagcgcgcg tgatagaaga aaagcactgggagaactaac agaggagaaa 2934 ggtgaaacac acacacattc ttaaggcaat aaaactagggggtgtatatt atcttctggt 2994 gcatgttctt ttctggaaaa tatggtagct cgccaaccgcatctgctcat ctgatattca 3054 aacacacagt attcgtgaat aagttgattc tgtcccccacgtggactctg tgctcaccca 3114 ttgtctcatt gccagtggtg tccaagggcc cccgttgggacccacggctc tcgtccctct 3174 gctccgtgtg tctcatgcca gcagcacgtc gccatccgtcaccagaatta gtcctcacag 3234 cctaggacca gttttgtatc aaactcgtct gatgttttgatgccatttgt cttttgtaaa 3294 gttaattcat taaaagtttt atgtactttg atttacagtgcctgtatctt ttattttcct 3354 gtcttctttc tcctgtggtt tgctccagaa ttaaggtttgttttccatcc attctccctt 3414 ttgacacagt tgtttcagaa agagctctcc agaagccaatattgagatgt aattcagatt 3474 aggacacagt gtgtgacgca gataactggt tactcagctccctggaaagc aggcaagcat 3534 gttgaatgta tctagtggtc tgattttaat ttgggcatctctagagaacg ctttcaggga 3594 aaaatacttt aatagtaaaa agattctctg cgagcaacagtgccccctcc gtccactacg 3654 ctcctgtctc caagaatgtt ttgctagagc taacagacatagactgcaaa agaataattt 3714 ggaatcagct atgcaaatca gtctcacaat agcgtgagctaactgagaga agtactaaga 3774 cccacaaact gcctgttaag tctgagaagg ctaaagaagacacacagcca acgttcatgc 3834 atttttaaag acagaaggcc ttgaagaatt tgttcttgtaaatccaacac aagttgtttg 3894 gtacttttaa cataaagaaa tcatactttg ccaaatagtgaaaagtagag caatcgtgta 3954 taagctaatg tttaaaagca aaactgcaaa ttgtagcccagttggtcaaa cttgttttct 4014 ttttataact catggcaggc atctgtaaga agtagagaacccagatgatc tcttaggaag 4074 ccttttattc gtgggaactc gaacttgaag cacaagttcctggtttgaat cctggctctg 4134 attttttact ggctgtgtga ctttgaacac atctcttagtccctcttagg agtactttcc 4194 ttattggcaa cttatggaat cgctagtgat taaacgaggcaatgactgtg agagagcctg 4254 gcaggtgccc cgtggtacat tcacagcacg ggcacagctgctgtgccagg actgtgactc 4314 attcccagta aaaggcactt atcgaagctg ataaccgtccttcatcaccg aagtgtgagt 4374 agagcatgac ttatttagta ttctgcctca atggggaattttttgatcct gtaatcacaa 4434 ctcagcattg gccttaatat acctaaatct ccaaaaacagtgattaaagc aagagaatta 4494 ttacaagggc ttttctcttt cctctaactc attcttcacggatgccgtag cgtttccgtg 4554 agctcaaact ggccttggtg taaaatgtgt aaggatgagcagcaggcgtg cctcgtgggt 4614 tcttcctctg ttacatcctg ctacactcat ctgcaggtcaccttagttca cctaccctga 4674 gtgaacaccc ccagctgggt ggtccaccaa gttctcataaacagagtccc tcccattccc 4734 ccacggggtg caccgaactt gggtttgcgc taaaaagaactcaaaaggag aactgtgctc 4794 tcccaaagcc atatcaccag tcttaccaaa caataggcttttaaaagcac tgagtcattg 4854 tcagaatcca ctatgggaag ctctgtgtgt acctggtcccttctaggtgt ggtcccatag 4914 gagcagcctt agcatcccct gtgaacttat caaaaatgcaaattctcagg ccccaacctg 4974 aaggaggacc ctgaatttga gatccccggg gctggggcccagcacaactg ctgtttagtg 5034 aacaggttct ccaggtgatt ctgatccctg atcaagcttcagacctccct ccctccccag 5094 tgtttttgca ggtgaggaaa caggggcaga gtaattcaggcacatgtgtt cagagttcca 5154 cagttgatta gcagttgagg ccaggctaga atggaaaactgcctgttcct tgaatctgaa 5214 agagcattat tcctggtcaa agctccttaa agttctgggcagctaaaagc atccctgtga 5274 gcaaaaatgc caggcagaaa actggcagtg cacctctcatcagcccaggt cccagtgcca 5334 ttggcttcaa gaaaaaaaaa aatctctccc caatgctctccttaaccttt aagtcttaca 5394 ggaagcctct catagaaatt gcctccagtc cagtttcccaaaaaccccag tgttttacat 5454 atctgtttag gagtgtctaa gttttgtcat caatccacgatgttattctt ccttcccaac 5514 tcactgtgct cctaaaggca gcaaccattc atctttcctttgctctggat acaccgaatg 5574 accaggtaac atcatcaggc cgggtgcagt ggctcctataatcccgatat tttgggaggc 5634 cgaggagaga ggatcactta agcccaggag tctgagaccagcctgggcaa catagcaaga 5694 cccccatctc tgcaaaaaaa taagaaaatt agcttggcatggtggcacgt gcctgtagtc 5754 ccagctacat gggaggctga ggtgggagga tcacttgagcccaggaagtc cagaacgtag 5814 tgagccttga ttataccact gcactccagc ctgggtgacagagcgagact ctgtctcag 5873 3 887 PRT Homo sapiens 3 Gly Ala Tyr His LysHis Leu Met Glu Leu Ala Leu Gln Gln Thr Tyr 1 5 10 15 Gln Asp Thr CysAsn Cys Ile Lys Ser Arg Ile Lys Leu Glu Phe Glu 20 25 30 Lys Arg Gln GlnGlu Arg Leu Leu Leu Ser Leu Leu Pro Ala His Ile 35 40 45 Ala Met Glu MetLys Ala Glu Ile Ile Gln Arg Leu Gln Gly Pro Lys 50 55 60 Ala Gly Gln MetGlu Asn Thr Asn Asn Phe His Asn Leu Tyr Val Lys 65 70 75 80 Arg His ThrAsn Val Ser Ile Leu Tyr Ala Asp Ile Val Gly Phe Thr 85 90 95 Arg Leu AlaSer Asp Cys Ser Pro Gly Glu Leu Val His Met Leu Asn 100 105 110 Glu LeuPhe Gly Lys Phe Asp Gln Ile Ala Lys Glu Asn Glu Cys Met 115 120 125 ArgIle Lys Ile Leu Gly Asp Cys Tyr Tyr Cys Val Ser Gly Leu Pro 130 135 140Ile Ser Leu Pro Asn His Ala Lys Asn Cys Val Lys Met Gly Leu Asp 145 150155 160 Met Cys Glu Ala Ile Lys Lys Val Arg Asp Ala Thr Gly Val Asp Ile165 170 175 Asn Met Arg Val Gly Val His Ser Gly Asn Val Leu Cys Gly ValIle 180 185 190 Gly Leu Gln Lys Trp Gln Tyr Asp Val Trp Ser His Asp ValThr Leu 195 200 205 Ala Asn His Met Glu Ala Gly Gly Val Pro Gly Arg ValHis Ile Ser 210 215 220 Ser Val Thr Leu Glu His Leu Asn Gly Ala Tyr LysVal Glu Glu Gly 225 230 235 240 Asp Gly Asp Ile Arg Asp Pro Tyr Leu LysGln His Leu Val Lys Thr 245 250 255 Tyr Phe Val Ile Asn Pro Lys Gly GluArg Arg Ser Pro Gln His Leu 260 265 270 Phe Arg Pro Arg His Thr Leu AspGly Ala Lys Met Arg Ala Ser Val 275 280 285 Arg Met Thr Arg Tyr Leu GluSer Trp Gly Ala Ala Lys Pro Phe Ala 290 295 300 His Leu His His Arg AspSer Met Thr Thr Glu Asn Gly Lys Ile Ser 305 310 315 320 Thr Thr Asp ValPro Met Gly Gln His Asn Phe Gln Asn Arg Thr Leu 325 330 335 Arg Thr LysSer Gln Lys Lys Arg Phe Glu Glu Glu Leu Asn Glu Arg 340 345 350 Met IleGln Ala Ile Asp Gly Ile Asn Ala Gln Lys Gln Trp Leu Lys 355 360 365 SerGlu Asp Ile Gln Arg Ile Ser Leu Leu Phe Tyr Asn Lys Val Leu 370 375 380Glu Lys Glu Tyr Arg Ala Thr Ala Leu Pro Ala Phe Lys Tyr Tyr Val 385 390395 400 Thr Cys Ala Cys Leu Ile Phe Phe Cys Ile Phe Ile Val Gln Ile Leu405 410 415 Val Leu Pro Lys Thr Ser Val Leu Gly Ile Ser Phe Gly Ala AlaPhe 420 425 430 Leu Leu Leu Ala Phe Ile Leu Phe Val Cys Phe Ala Gly GlnLeu Leu 435 440 445 Gln Cys Ser Lys Lys Ala Ser Pro Leu Leu Met Trp LeuLeu Lys Ser 450 455 460 Ser Gly Ile Ile Ala Asn Arg Pro Trp Pro Arg IleSer Leu Thr Ile 465 470 475 480 Ile Thr Thr Ala Ile Ile Leu Met Met AlaVal Phe Asn Met Phe Phe 485 490 495 Leu Ser Asp Ser Glu Glu Thr Ile ProPro Thr Ala Asn Thr Thr Asn 500 505 510 Thr Ser Phe Ser Ala Ser Asn AsnGln Val Ala Ile Leu Arg Ala Gln 515 520 525 Asn Leu Phe Phe Leu Pro TyrPhe Ile Tyr Ser Cys Ile Leu Gly Leu 530 535 540 Ile Ser Cys Ser Val PheLeu Arg Val Asn Tyr Glu Leu Lys Met Leu 545 550 555 560 Ile Met Met ValAla Leu Val Gly Tyr Asn Thr Ile Leu Leu His Thr 565 570 575 His Ala HisVal Leu Gly Asp Tyr Ser Gln Val Leu Phe Glu Arg Pro 580 585 590 Gly IleTrp Lys Asp Leu Lys Thr Met Gly Ser Val Ser Leu Ser Ile 595 600 605 PhePhe Ile Thr Leu Leu Val Leu Gly Arg Gln Asn Glu Tyr Tyr Cys 610 615 620Arg Leu Asp Phe Leu Trp Lys Asn Lys Phe Lys Lys Glu Arg Glu Glu 625 630635 640 Ile Glu Thr Met Glu Asn Leu Asn Arg Val Leu Leu Glu Asn Val Leu645 650 655 Pro Ala His Val Ala Glu His Phe Leu Ala Arg Ser Leu Lys AsnGlu 660 665 670 Glu Leu Tyr His Gln Ser Tyr Asp Cys Val Cys Val Met PheAla Ser 675 680 685 Ile Pro Asp Phe Lys Glu Phe Tyr Thr Glu Ser Asp ValAsn Lys Glu 690 695 700 Gly Leu Glu Cys Leu Arg Leu Leu Asn Glu Ile IleAla Asp Phe Asp 705 710 715 720 Asp Leu Leu Ser Lys Pro Lys Phe Ser GlyVal Glu Lys Ile Lys Thr 725 730 735 Ile Gly Ser Thr Tyr Met Ala Ala ThrGly Leu Ser Ala Val Pro Ser 740 745 750 Gln Glu His Ser Gln Glu Pro GluArg Gln Tyr Met His Ile Gly Thr 755 760 765 Met Val Glu Phe Ala Phe AlaLeu Val Gly Lys Leu Asp Ala Ile Asn 770 775 780 Lys His Ser Phe Asn AspPhe Lys Leu Arg Val Gly Ile Asn His Gly 785 790 795 800 Pro Val Ile AlaGly Val Ile Gly Ala Gln Lys Pro Gln Tyr Asp Ile 805 810 815 Trp Gly AsnThr Val Asn Val Ala Ser Arg Met Asp Ser Thr Gly Val 820 825 830 Leu AspLys Ile Gln Val Thr Glu Glu Thr Ser Leu Val Leu Gln Thr 835 840 845 LeuGly Tyr Thr Cys Thr Cys Arg Gly Ile Ile Asn Val Lys Gly Lys 850 855 860Gly Asp Leu Lys Thr Tyr Phe Val Asn Thr Glu Met Ser Arg Ser Leu 865 870875 880 Ser Gln Ser Asn Val Ala Ser 885 4 3261 DNA Homo sapiens 4atgcggcacc gccgctacct gcgggaccgc tccgaggagg cggcgggcgg cggagacggg 60ctgccgcggt cccgggactg gctctacgag tcctactact gcatgagcca gcagcacccg 120ctcatcgtct tcctgctgct catcgtcatg ggctcctgcc tcgccctgct cgccgtcttc 180ttcgcgctcg ggctggaagt tgaagaccat gtggcgtttc taataacagt tccaactgcc 240ctggcgattt tctttgcgat atttatcctg gtctgcatcg agtctgtgtt taagaagctg 300ctgcgcctct tctcgttggt gatatggata tgccttgttg ccatgggata cctgttcatg 360tgttttggag gcaccgtctc tccctgggac caggtatcgt tcttcctctt catcatcttc 420gtggtgtaca ccatgctgcc cttcaacatg cgagacgcca tcattgccag cgtcctcacc 480tcctcctccc acaccatcgt gcttagcgtc tgcctgtctg caacaccggg aggcaaggag 540cacctggtct ggcagatcct ggccaatgtg atcattttca tctgtgggaa cctggcggga 600gcctaccata agcacctcat ggaactcgct cttcagcaaa catatcagga cacctgtaat 660tgcatcaagt cgcggatcaa gttggaattt gaaaaacgtc aacaggagcg gcttctgctc 720tccctgctgc cggcccacat cgccatggag atgaaagcgg agatcatcca gaggctgcag 780ggccccaagg cgggccagat ggagaacaca aataacttcc acaacctgta tgtgaagcgg 840catacaaacg tgagcatctt atacgctgac atcgttggct ttacccggct ggcaagtgac 900tgctccccgg gagaactagt ccacatgctg aatgagctct ttggaaagtt tgatcaaatt 960gcaaaggaga atgaatgcat gagaattaaa attttaggag actgctacta ctgtgtatct 1020ggactcccta tatctctccc taaccatgcc aagaactgtg tgaaaatggg gctggacatg 1080tgtgaagcca taaagaaagt gagggatgct actggagttg atatcaacat gcgcgtgggc 1140gtgcattctg ggaatgtcct gtgtggcgtg attggtctgc agaagtggca atatgatgtg 1200tggtcacatg atgtgacctt ggccaaccac atggaagctg gaggggtccc tggacgtgtt 1260cacatttctt ctgtcaccct ggagcacttg aatggcgctt ataaagtgga ggagggagat 1320ggtgacatta gggacccata tttaaaacag cacctggtga aaacctactt tgtgatcaac 1380cccaagggag aacgacggag cccccagcat ctcttcagac ctcgccacac ccttgatgga 1440gccaaaatga gggcctcggt ccgcatgacc cggtacttgg agtcctgggg ggcagccaag 1500ccctttgcac acctacatca cagggacagc atgaccacag agaacggcaa gatcagcacc 1560acggatgtac ccatgggtca gcataatttt caaaatcgca ccttaagaac caagtcacaa 1620aagaagagat ttgaagaaga attgaatgaa aggatgattc aagcaattga tgggattaat 1680gcacagaagc aatggctcaa gtctgaagac attcagagaa tctcactgct tttctataac 1740aaagtactag aaaaagagta ccgggccacg gcactgccag cgttcaagta ttatgtgact 1800tgtgcctgtc tcatattctt ctgcatcttc attgtgcaga ttctcgtgct gccaaaaacg 1860tctgtcctgg gcatctcctt tggggctgcg tttctcttgc tggccttcat cctcttcgtc 1920tgctttgctg gacagcttct gcaatgcagc aaaaaagcct ctcccctgct catgtggctt 1980ttgaagtcct cgggcatcat tgccaaccgc ccctggccac ggatctctct cacgatcatc 2040accacagcca tcatattaat gatggccgtg ttcaacatgt ttttcctgag tgactcagag 2100gaaacaatcc ctccaactgc caacacaaca aacacaagct tttcagcctc aaataatcag 2160gtggcgattc tgcgtgcgca gaatttattt ttcctcccgt actttatcta cagctgcatt 2220ctgggactga tatcctgttc cgtgttcctg cgggtaaact atgagctgaa gatgttgatc 2280atgatggtgg ccttggtggg ctacaacacc atcctactcc acacccacgc ccacgtcctg 2340ggcgactaca gccaggtctt atttgagaga ccaggcattt ggaaagacct gaagaccatg 2400ggctctgtgt ctctctctat attcttcatc acactgcttg ttctgggtag acagaatgaa 2460tattactgta ggttagactt cttatggaag aacaaattca aaaaagagcg ggaggagata 2520gagaccatgg agaacctgaa ccgcgtgctg ctggagaacg tgcttcccgc gcacgtggct 2580gagcacttcc tggccaggag cctgaagaat gaggagctat accaccagtc ctatgactgc 2640gtctgtgtca tgtttgcctc cattccggat ttcaaagaat tttatacaga atccgacgtg 2700aacaaggagg gcttggaatg ccttcggctc ctgaacgaga tcatcgctga ctttgatgat 2760cttctttcca agccaaaatt cagtggagtt gaaaagatta agaccattgg cagcacatac 2820atggcagcaa caggtctgag cgctgtgccc agccaggagc actcccagga gcccgagcgg 2880cagtacatgc acattggcac catggtggag tttgcttttg ccctggtagg gaagctggat 2940gccatcaaca agcactcctt caacgacttc aaattgcgag tgggtattaa ccatggacct 3000gtgatagctg gtgtgattgg agctcagaag ccacaatatg atatctgggg caacactgtc 3060aatgtggcca gtaggatgga cagcaccgga gtcctggaca aaatacaggt taccgaggag 3120acgagcctcg tcctgcagac cctcggatac acgtgcacct gtcgaggaat aatcaacgtg 3180aaaggaaagg gggacctgaa gacgtacttt gtaaacacag aaatgtcaag gtccctttcc 3240cagagcaacg tggcatcctg a 3261 5 6470 DNA Homo sapiens CDS (1)..(3261) 5atg cgg cac cgc cgc tac ctg cgg gac cgc tcc gag gag gcg gcg ggc 48 MetArg His Arg Arg Tyr Leu Arg Asp Arg Ser Glu Glu Ala Ala Gly 1 5 10 15ggc gga gac ggg ctg ccg cgg tcc cgg gac tgg ctc tac gag tcc tac 96 GlyGly Asp Gly Leu Pro Arg Ser Arg Asp Trp Leu Tyr Glu Ser Tyr 20 25 30 tactgc atg agc cag cag cac ccg ctc atc gtc ttc ctg ctg ctc atc 144 Tyr CysMet Ser Gln Gln His Pro Leu Ile Val Phe Leu Leu Leu Ile 35 40 45 gtc atgggc tcc tgc ctc gcc ctg ctc gcc gtc ttc ttc gcg ctc ggg 192 Val Met GlySer Cys Leu Ala Leu Leu Ala Val Phe Phe Ala Leu Gly 50 55 60 ctg gaa gttgaa gac cat gtg gcg ttt cta ata aca gtt cca act gcc 240 Leu Glu Val GluAsp His Val Ala Phe Leu Ile Thr Val Pro Thr Ala 65 70 75 80 ctg gcg attttc ttt gcg ata ttt atc ctg gtc tgc atc gag tct gtg 288 Leu Ala Ile PhePhe Ala Ile Phe Ile Leu Val Cys Ile Glu Ser Val 85 90 95 ttt aag aag ctgctg cgc ctc ttc tcg ttg gtg ata tgg ata tgc ctt 336 Phe Lys Lys Leu LeuArg Leu Phe Ser Leu Val Ile Trp Ile Cys Leu 100 105 110 gtt gcc atg ggatac ctg ttc atg tgt ttt gga ggc acc gtc tct ccc 384 Val Ala Met Gly TyrLeu Phe Met Cys Phe Gly Gly Thr Val Ser Pro 115 120 125 tgg gac cag gtatcg ttc ttc ctc ttc atc atc ttc gtg gtg tac acc 432 Trp Asp Gln Val SerPhe Phe Leu Phe Ile Ile Phe Val Val Tyr Thr 130 135 140 atg ctg ccc ttcaac atg cga gac gcc atc att gcc agc gtc ctc acc 480 Met Leu Pro Phe AsnMet Arg Asp Ala Ile Ile Ala Ser Val Leu Thr 145 150 155 160 tcc tcc tcccac acc atc gtg ctt agc gtc tgc ctg tct gca aca ccg 528 Ser Ser Ser HisThr Ile Val Leu Ser Val Cys Leu Ser Ala Thr Pro 165 170 175 gga ggc aaggag cac ctg gtc tgg cag atc ctg gcc aat gtg atc att 576 Gly Gly Lys GluHis Leu Val Trp Gln Ile Leu Ala Asn Val Ile Ile 180 185 190 ttc atc tgtggg aac ctg gcg gga gcc tac cat aag cac ctc atg gaa 624 Phe Ile Cys GlyAsn Leu Ala Gly Ala Tyr His Lys His Leu Met Glu 195 200 205 ctc gct cttcag caa aca tat cag gac acc tgt aat tgc atc aag tcg 672 Leu Ala Leu GlnGln Thr Tyr Gln Asp Thr Cys Asn Cys Ile Lys Ser 210 215 220 cgg atc aagttg gaa ttt gaa aaa cgt caa cag gag cgg ctt ctg ctc 720 Arg Ile Lys LeuGlu Phe Glu Lys Arg Gln Gln Glu Arg Leu Leu Leu 225 230 235 240 tcc ctgctg ccg gcc cac atc gcc atg gag atg aaa gcg gag atc atc 768 Ser Leu LeuPro Ala His Ile Ala Met Glu Met Lys Ala Glu Ile Ile 245 250 255 cag aggctg cag ggc ccc aag gcg ggc cag atg gag aac aca aat aac 816 Gln Arg LeuGln Gly Pro Lys Ala Gly Gln Met Glu Asn Thr Asn Asn 260 265 270 ttc cacaac ctg tat gtg aag cgg cat aca aac gtg agc atc tta tac 864 Phe His AsnLeu Tyr Val Lys Arg His Thr Asn Val Ser Ile Leu Tyr 275 280 285 gct gacatc gtt ggc ttt acc cgg ctg gca agt gac tgc tcc ccg gga 912 Ala Asp IleVal Gly Phe Thr Arg Leu Ala Ser Asp Cys Ser Pro Gly 290 295 300 gaa ctagtc cac atg ctg aat gag ctc ttt gga aag ttt gat caa att 960 Glu Leu ValHis Met Leu Asn Glu Leu Phe Gly Lys Phe Asp Gln Ile 305 310 315 320 gcaaag gag aat gaa tgc atg aga att aaa att tta gga gac tgc tac 1008 Ala LysGlu Asn Glu Cys Met Arg Ile Lys Ile Leu Gly Asp Cys Tyr 325 330 335 tactgt gta tct gga ctc cct ata tct ctc cct aac cat gcc aag aac 1056 Tyr CysVal Ser Gly Leu Pro Ile Ser Leu Pro Asn His Ala Lys Asn 340 345 350 tgtgtg aaa atg ggg ctg gac atg tgt gaa gcc ata aag aaa gtg agg 1104 Cys ValLys Met Gly Leu Asp Met Cys Glu Ala Ile Lys Lys Val Arg 355 360 365 gatgct act gga gtt gat atc aac atg cgc gtg ggc gtg cat tct ggg 1152 Asp AlaThr Gly Val Asp Ile Asn Met Arg Val Gly Val His Ser Gly 370 375 380 aatgtc ctg tgt ggc gtg att ggt ctg cag aag tgg caa tat gat gtg 1200 Asn ValLeu Cys Gly Val Ile Gly Leu Gln Lys Trp Gln Tyr Asp Val 385 390 395 400tgg tca cat gat gtg acc ttg gcc aac cac atg gaa gct gga ggg gtc 1248 TrpSer His Asp Val Thr Leu Ala Asn His Met Glu Ala Gly Gly Val 405 410 415cct gga cgt gtt cac att tct tct gtc acc ctg gag cac ttg aat ggc 1296 ProGly Arg Val His Ile Ser Ser Val Thr Leu Glu His Leu Asn Gly 420 425 430gct tat aaa gtg gag gag gga gat ggt gac att agg gac cca tat tta 1344 AlaTyr Lys Val Glu Glu Gly Asp Gly Asp Ile Arg Asp Pro Tyr Leu 435 440 445aaa cag cac ctg gtg aaa acc tac ttt gtg atc aac ccc aag gga gaa 1392 LysGln His Leu Val Lys Thr Tyr Phe Val Ile Asn Pro Lys Gly Glu 450 455 460cga cgg agc ccc cag cat ctc ttc aga cct cgc cac acc ctt gat gga 1440 ArgArg Ser Pro Gln His Leu Phe Arg Pro Arg His Thr Leu Asp Gly 465 470 475480 gcc aaa atg agg gcc tcg gtc cgc atg acc cgg tac ttg gag tcc tgg 1488Ala Lys Met Arg Ala Ser Val Arg Met Thr Arg Tyr Leu Glu Ser Trp 485 490495 ggg gca gcc aag ccc ttt gca cac cta cat cac agg gac agc atg acc 1536Gly Ala Ala Lys Pro Phe Ala His Leu His His Arg Asp Ser Met Thr 500 505510 aca gag aac ggc aag atc agc acc acg gat gta ccc atg ggt cag cat 1584Thr Glu Asn Gly Lys Ile Ser Thr Thr Asp Val Pro Met Gly Gln His 515 520525 aat ttt caa aat cgc acc tta aga acc aag tca caa aag aag aga ttt 1632Asn Phe Gln Asn Arg Thr Leu Arg Thr Lys Ser Gln Lys Lys Arg Phe 530 535540 gaa gaa gaa ttg aat gaa agg atg att caa gca att gat ggg att aat 1680Glu Glu Glu Leu Asn Glu Arg Met Ile Gln Ala Ile Asp Gly Ile Asn 545 550555 560 gca cag aag caa tgg ctc aag tct gaa gac att cag aga atc tca ctg1728 Ala Gln Lys Gln Trp Leu Lys Ser Glu Asp Ile Gln Arg Ile Ser Leu 565570 575 ctt ttc tat aac aaa gta cta gaa aaa gag tac cgg gcc acg gca ctg1776 Leu Phe Tyr Asn Lys Val Leu Glu Lys Glu Tyr Arg Ala Thr Ala Leu 580585 590 cca gcg ttc aag tat tat gtg act tgt gcc tgt ctc ata ttc ttc tgc1824 Pro Ala Phe Lys Tyr Tyr Val Thr Cys Ala Cys Leu Ile Phe Phe Cys 595600 605 atc ttc att gtg cag att ctc gtg ctg cca aaa acg tct gtc ctg ggc1872 Ile Phe Ile Val Gln Ile Leu Val Leu Pro Lys Thr Ser Val Leu Gly 610615 620 atc tcc ttt ggg gct gcg ttt ctc ttg ctg gcc ttc atc ctc ttc gtc1920 Ile Ser Phe Gly Ala Ala Phe Leu Leu Leu Ala Phe Ile Leu Phe Val 625630 635 640 tgc ttt gct gga cag ctt ctg caa tgc agc aaa aaa gcc tct cccctg 1968 Cys Phe Ala Gly Gln Leu Leu Gln Cys Ser Lys Lys Ala Ser Pro Leu645 650 655 ctc atg tgg ctt ttg aag tcc tcg ggc atc att gcc aac cgc ccctgg 2016 Leu Met Trp Leu Leu Lys Ser Ser Gly Ile Ile Ala Asn Arg Pro Trp660 665 670 cca cgg atc tct ctc acg atc atc acc aca gcc atc ata tta atgatg 2064 Pro Arg Ile Ser Leu Thr Ile Ile Thr Thr Ala Ile Ile Leu Met Met675 680 685 gcc gtg ttc aac atg ttt ttc ctg agt gac tca gag gaa aca atccct 2112 Ala Val Phe Asn Met Phe Phe Leu Ser Asp Ser Glu Glu Thr Ile Pro690 695 700 cca act gcc aac aca aca aac aca agc ttt tca gcc tca aat aatcag 2160 Pro Thr Ala Asn Thr Thr Asn Thr Ser Phe Ser Ala Ser Asn Asn Gln705 710 715 720 gtg gcg att ctg cgt gcg cag aat tta ttt ttc ctc ccg tacttt atc 2208 Val Ala Ile Leu Arg Ala Gln Asn Leu Phe Phe Leu Pro Tyr PheIle 725 730 735 tac agc tgc att ctg gga ctg ata tcc tgt tcc gtg ttc ctgcgg gta 2256 Tyr Ser Cys Ile Leu Gly Leu Ile Ser Cys Ser Val Phe Leu ArgVal 740 745 750 aac tat gag ctg aag atg ttg atc atg atg gtg gcc ttg gtgggc tac 2304 Asn Tyr Glu Leu Lys Met Leu Ile Met Met Val Ala Leu Val GlyTyr 755 760 765 aac acc atc cta ctc cac acc cac gcc cac gtc ctg ggc gactac agc 2352 Asn Thr Ile Leu Leu His Thr His Ala His Val Leu Gly Asp TyrSer 770 775 780 cag gtc tta ttt gag aga cca ggc att tgg aaa gac ctg aagacc atg 2400 Gln Val Leu Phe Glu Arg Pro Gly Ile Trp Lys Asp Leu Lys ThrMet 785 790 795 800 ggc tct gtg tct ctc tct ata ttc ttc atc aca ctg cttgtt ctg ggt 2448 Gly Ser Val Ser Leu Ser Ile Phe Phe Ile Thr Leu Leu ValLeu Gly 805 810 815 aga cag aat gaa tat tac tgt agg tta gac ttc tta tggaag aac aaa 2496 Arg Gln Asn Glu Tyr Tyr Cys Arg Leu Asp Phe Leu Trp LysAsn Lys 820 825 830 ttc aaa aaa gag cgg gag gag ata gag acc atg gag aacctg aac cgc 2544 Phe Lys Lys Glu Arg Glu Glu Ile Glu Thr Met Glu Asn LeuAsn Arg 835 840 845 gtg ctg ctg gag aac gtg ctt ccc gcg cac gtg gct gagcac ttc ctg 2592 Val Leu Leu Glu Asn Val Leu Pro Ala His Val Ala Glu HisPhe Leu 850 855 860 gcc agg agc ctg aag aat gag gag cta tac cac cag tcctat gac tgc 2640 Ala Arg Ser Leu Lys Asn Glu Glu Leu Tyr His Gln Ser TyrAsp Cys 865 870 875 880 gtc tgt gtc atg ttt gcc tcc att ccg gat ttc aaagaa ttt tat aca 2688 Val Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys GluPhe Tyr Thr 885 890 895 gaa tcc gac gtg aac aag gag ggc ttg gaa tgc cttcgg ctc ctg aac 2736 Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu ArgLeu Leu Asn 900 905 910 gag atc atc gct gac ttt gat gat ctt ctt tcc aagcca aaa ttc agt 2784 Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys ProLys Phe Ser 915 920 925 gga gtt gaa aag att aag acc att ggc agc aca tacatg gca gca aca 2832 Gly Val Glu Lys Ile Lys Thr Ile Gly Ser Thr Tyr MetAla Ala Thr 930 935 940 ggt ctg agc gct gtg ccc agc cag gag cac tcc caggag ccc gag cgg 2880 Gly Leu Ser Ala Val Pro Ser Gln Glu His Ser Gln GluPro Glu Arg 945 950 955 960 cag tac atg cac att ggc acc atg gtg gag tttgct ttt gcc ctg gta 2928 Gln Tyr Met His Ile Gly Thr Met Val Glu Phe AlaPhe Ala Leu Val 965 970 975 ggg aag ctg gat gcc atc aac aag cac tcc ttcaac gac ttc aaa ttg 2976 Gly Lys Leu Asp Ala Ile Asn Lys His Ser Phe AsnAsp Phe Lys Leu 980 985 990 cga gtg ggt att aac cat gga cct gtg ata gctggt gtg att gga gct 3024 Arg Val Gly Ile Asn His Gly Pro Val Ile Ala GlyVal Ile Gly Ala 995 1000 1005 cag aag cca caa tat gat atc tgg ggc aacact gtc aat gtg gcc 3069 Gln Lys Pro Gln Tyr Asp Ile Trp Gly Asn Thr ValAsn Val Ala 1010 1015 1020 agt agg atg gac agc acc gga gtc ctg gac aaaata cag gtt acc 3114 Ser Arg Met Asp Ser Thr Gly Val Leu Asp Lys Ile GlnVal Thr 1025 1030 1035 gag gag acg agc ctc gtc ctg cag acc ctc gga tacacg tgc acc 3159 Glu Glu Thr Ser Leu Val Leu Gln Thr Leu Gly Tyr Thr CysThr 1040 1045 1050 tgt cga gga ata atc aac gtg aaa gga aag ggg gac ctgaag acg 3204 Cys Arg Gly Ile Ile Asn Val Lys Gly Lys Gly Asp Leu Lys Thr1055 1060 1065 tac ttt gta aac aca gaa atg tca agg tcc ctt tcc cag agcaac 3249 Tyr Phe Val Asn Thr Glu Met Ser Arg Ser Leu Ser Gln Ser Asn1070 1075 1080 gtg gca tcc tga agagtcacct tcattttggc aagaagactgtattttcagg 3301 Val Ala Ser 1085 aaggtatcac acactttctg actgcaacttctgtcccttg tttttgatgt gcgtgctgtc 3361 tgtcctatgg agcctctgca gactcgttctcgtgacccag tggcataccg tttggtgtct 3421 gatgtgtgcc cagatcgttc tgccacttgcactgtgcttg ctcctaagca aaagggaaaa 3481 ggagcgcgcg tgatagaaga aaagcactgggagaactaac agaggagaaa ggtgaaacac 3541 acacacattc ttaaggcaat aaaactagggggtgtatatt atcttctggt gcatgttctt 3601 ttctggaaaa tatggtagct cgccaaccgcatctgctcat ctgatattca aacacacagt 3661 attcgtgaat aagttgattc tgtcccccacgtggactctg tgctcaccca ttgtctcatt 3721 gccagtggtg tccaagggcc cccgttgggacccacggctc tcgtccctct gctccgtgtg 3781 tctcatgcca gcagcacgtc gccatccgtcaccagaatta gtcctcacag cctaggacca 3841 gttttgtatc aaactcgtct gatgttttgatgccatttgt cttttgtaaa gttaattcat 3901 taaaagtttt atgtactttg atttacagtgcctgtatctt ttattttcct gtcttctttc 3961 tcctgtggtt tgctccagaa ttaaggtttgttttccatcc attctccctt ttgacacagt 4021 tgtttcagaa agagctctcc agaagccaatattgagatgt aattcagatt aggacacagt 4081 gtgtgacgca gataactggt tactcagctccctggaaagc aggcaagcat gttgaatgta 4141 tctagtggtc tgattttaat ttgggcatctctagagaacg ctttcaggga aaaatacttt 4201 aatagtaaaa agattctctg cgagcaacagtgccccctcc gtccactacg ctcctgtctc 4261 caagaatgtt ttgctagagc taacagacatagactgcaaa agaataattt ggaatcagct 4321 atgcaaatca gtctcacaat agcgtgagctaactgagaga agtactaaga cccacaaact 4381 gcctgttaag tctgagaagg ctaaagaagacacacagcca acgttcatgc atttttaaag 4441 acagaaggcc ttgaagaatt tgttcttgtaaatccaacac aagttgtttg gtacttttaa 4501 cataaagaaa tcatactttg ccaaatagtgaaaagtagag caatcgtgta taagctaatg 4561 tttaaaagca aaactgcaaa ttgtagcccagttggtcaaa cttgttttct ttttataact 4621 catggcaggc atctgtaaga agtagagaacccagatgatc tcttaggaag ccttttattc 4681 gtgggaactc gaacttgaag cacaagttcctggtttgaat cctggctctg attttttact 4741 ggctgtgtga ctttgaacac atctcttagtccctcttagg agtactttcc ttattggcaa 4801 cttatggaat cgctagtgat taaacgaggcaatgactgtg agagagcctg gcaggtgccc 4861 cgtggtacat tcacagcacg ggcacagctgctgtgccagg actgtgactc attcccagta 4921 aaaggcactt atcgaagctg ataaccgtccttcatcaccg aagtgtgagt agagcatgac 4981 ttatttagta ttctgcctca atggggaattttttgatcct gtaatcacaa ctcagcattg 5041 gccttaatat acctaaatct ccaaaaacagtgattaaagc aagagaatta ttacaagggc 5101 ttttctcttt cctctaactc attcttcacggatgccgtag cgtttccgtg agctcaaact 5161 ggccttggtg taaaatgtgt aaggatgagcagcaggcgtg cctcgtgggt tcttcctctg 5221 ttacatcctg ctacactcat ctgcaggtcaccttagttca cctaccctga gtgaacaccc 5281 ccagctgggt ggtccaccaa gttctcataaacagagtccc tcccattccc ccacggggtg 5341 caccgaactt gggtttgcgc taaaaagaactcaaaaggag aactgtgctc tcccaaagcc 5401 atatcaccag tcttaccaaa caataggcttttaaaagcac tgagtcattg tcagaatcca 5461 ctatgggaag ctctgtgtgt acctggtcccttctaggtgt ggtcccatag gagcagcctt 5521 agcatcccct gtgaacttat caaaaatgcaaattctcagg ccccaacctg aaggaggacc 5581 ctgaatttga gatccccggg gctggggcccagcacaactg ctgtttagtg aacaggttct 5641 ccaggtgatt ctgatccctg atcaagcttcagacctccct ccctccccag tgtttttgca 5701 ggtgaggaaa caggggcaga gtaattcaggcacatgtgtt cagagttcca cagttgatta 5761 gcagttgagg ccaggctaga atggaaaactgcctgttcct tgaatctgaa agagcattat 5821 tcctggtcaa agctccttaa agttctgggcagctaaaagc atccctgtga gcaaaaatgc 5881 caggcagaaa actggcagtg cacctctcatcagcccaggt cccagtgcca ttggcttcaa 5941 gaaaaaaaaa aatctctccc caatgctctccttaaccttt aagtcttaca ggaagcctct 6001 catagaaatt gcctccagtc cagtttcccaaaaaccccag tgttttacat atctgtttag 6061 gagtgtctaa gttttgtcat caatccacgatgttattctt ccttcccaac tcactgtgct 6121 cctaaaggca gcaaccattc atctttcctttgctctggat acaccgaatg accaggtaac 6181 atcatcaggc cgggtgcagt ggctcctataatcccgatat tttgggaggc cgaggagaga 6241 ggatcactta agcccaggag tctgagaccagcctgggcaa catagcaaga cccccatctc 6301 tgcaaaaaaa taagaaaatt agcttggcatggtggcacgt gcctgtagtc ccagctacat 6361 gggaggctga ggtgggagga tcacttgagcccaggaagtc cagaacgtag tgagccttga 6421 ttataccact gcactccagc ctgggtgacagagcgagact ctgtctcag 6470 6 1086 PRT Homo sapiens 6 Met Arg His Arg ArgTyr Leu Arg Asp Arg Ser Glu Glu Ala Ala Gly 1 5 10 15 Gly Gly Asp GlyLeu Pro Arg Ser Arg Asp Trp Leu Tyr Glu Ser Tyr 20 25 30 Tyr Cys Met SerGln Gln His Pro Leu Ile Val Phe Leu Leu Leu Ile 35 40 45 Val Met Gly SerCys Leu Ala Leu Leu Ala Val Phe Phe Ala Leu Gly 50 55 60 Leu Glu Val GluAsp His Val Ala Phe Leu Ile Thr Val Pro Thr Ala 65 70 75 80 Leu Ala IlePhe Phe Ala Ile Phe Ile Leu Val Cys Ile Glu Ser Val 85 90 95 Phe Lys LysLeu Leu Arg Leu Phe Ser Leu Val Ile Trp Ile Cys Leu 100 105 110 Val AlaMet Gly Tyr Leu Phe Met Cys Phe Gly Gly Thr Val Ser Pro 115 120 125 TrpAsp Gln Val Ser Phe Phe Leu Phe Ile Ile Phe Val Val Tyr Thr 130 135 140Met Leu Pro Phe Asn Met Arg Asp Ala Ile Ile Ala Ser Val Leu Thr 145 150155 160 Ser Ser Ser His Thr Ile Val Leu Ser Val Cys Leu Ser Ala Thr Pro165 170 175 Gly Gly Lys Glu His Leu Val Trp Gln Ile Leu Ala Asn Val IleIle 180 185 190 Phe Ile Cys Gly Asn Leu Ala Gly Ala Tyr His Lys His LeuMet Glu 195 200 205 Leu Ala Leu Gln Gln Thr Tyr Gln Asp Thr Cys Asn CysIle Lys Ser 210 215 220 Arg Ile Lys Leu Glu Phe Glu Lys Arg Gln Gln GluArg Leu Leu Leu 225 230 235 240 Ser Leu Leu Pro Ala His Ile Ala Met GluMet Lys Ala Glu Ile Ile 245 250 255 Gln Arg Leu Gln Gly Pro Lys Ala GlyGln Met Glu Asn Thr Asn Asn 260 265 270 Phe His Asn Leu Tyr Val Lys ArgHis Thr Asn Val Ser Ile Leu Tyr 275 280 285 Ala Asp Ile Val Gly Phe ThrArg Leu Ala Ser Asp Cys Ser Pro Gly 290 295 300 Glu Leu Val His Met LeuAsn Glu Leu Phe Gly Lys Phe Asp Gln Ile 305 310 315 320 Ala Lys Glu AsnGlu Cys Met Arg Ile Lys Ile Leu Gly Asp Cys Tyr 325 330 335 Tyr Cys ValSer Gly Leu Pro Ile Ser Leu Pro Asn His Ala Lys Asn 340 345 350 Cys ValLys Met Gly Leu Asp Met Cys Glu Ala Ile Lys Lys Val Arg 355 360 365 AspAla Thr Gly Val Asp Ile Asn Met Arg Val Gly Val His Ser Gly 370 375 380Asn Val Leu Cys Gly Val Ile Gly Leu Gln Lys Trp Gln Tyr Asp Val 385 390395 400 Trp Ser His Asp Val Thr Leu Ala Asn His Met Glu Ala Gly Gly Val405 410 415 Pro Gly Arg Val His Ile Ser Ser Val Thr Leu Glu His Leu AsnGly 420 425 430 Ala Tyr Lys Val Glu Glu Gly Asp Gly Asp Ile Arg Asp ProTyr Leu 435 440 445 Lys Gln His Leu Val Lys Thr Tyr Phe Val Ile Asn ProLys Gly Glu 450 455 460 Arg Arg Ser Pro Gln His Leu Phe Arg Pro Arg HisThr Leu Asp Gly 465 470 475 480 Ala Lys Met Arg Ala Ser Val Arg Met ThrArg Tyr Leu Glu Ser Trp 485 490 495 Gly Ala Ala Lys Pro Phe Ala His LeuHis His Arg Asp Ser Met Thr 500 505 510 Thr Glu Asn Gly Lys Ile Ser ThrThr Asp Val Pro Met Gly Gln His 515 520 525 Asn Phe Gln Asn Arg Thr LeuArg Thr Lys Ser Gln Lys Lys Arg Phe 530 535 540 Glu Glu Glu Leu Asn GluArg Met Ile Gln Ala Ile Asp Gly Ile Asn 545 550 555 560 Ala Gln Lys GlnTrp Leu Lys Ser Glu Asp Ile Gln Arg Ile Ser Leu 565 570 575 Leu Phe TyrAsn Lys Val Leu Glu Lys Glu Tyr Arg Ala Thr Ala Leu 580 585 590 Pro AlaPhe Lys Tyr Tyr Val Thr Cys Ala Cys Leu Ile Phe Phe Cys 595 600 605 IlePhe Ile Val Gln Ile Leu Val Leu Pro Lys Thr Ser Val Leu Gly 610 615 620Ile Ser Phe Gly Ala Ala Phe Leu Leu Leu Ala Phe Ile Leu Phe Val 625 630635 640 Cys Phe Ala Gly Gln Leu Leu Gln Cys Ser Lys Lys Ala Ser Pro Leu645 650 655 Leu Met Trp Leu Leu Lys Ser Ser Gly Ile Ile Ala Asn Arg ProTrp 660 665 670 Pro Arg Ile Ser Leu Thr Ile Ile Thr Thr Ala Ile Ile LeuMet Met 675 680 685 Ala Val Phe Asn Met Phe Phe Leu Ser Asp Ser Glu GluThr Ile Pro 690 695 700 Pro Thr Ala Asn Thr Thr Asn Thr Ser Phe Ser AlaSer Asn Asn Gln 705 710 715 720 Val Ala Ile Leu Arg Ala Gln Asn Leu PhePhe Leu Pro Tyr Phe Ile 725 730 735 Tyr Ser Cys Ile Leu Gly Leu Ile SerCys Ser Val Phe Leu Arg Val 740 745 750 Asn Tyr Glu Leu Lys Met Leu IleMet Met Val Ala Leu Val Gly Tyr 755 760 765 Asn Thr Ile Leu Leu His ThrHis Ala His Val Leu Gly Asp Tyr Ser 770 775 780 Gln Val Leu Phe Glu ArgPro Gly Ile Trp Lys Asp Leu Lys Thr Met 785 790 795 800 Gly Ser Val SerLeu Ser Ile Phe Phe Ile Thr Leu Leu Val Leu Gly 805 810 815 Arg Gln AsnGlu Tyr Tyr Cys Arg Leu Asp Phe Leu Trp Lys Asn Lys 820 825 830 Phe LysLys Glu Arg Glu Glu Ile Glu Thr Met Glu Asn Leu Asn Arg 835 840 845 ValLeu Leu Glu Asn Val Leu Pro Ala His Val Ala Glu His Phe Leu 850 855 860Ala Arg Ser Leu Lys Asn Glu Glu Leu Tyr His Gln Ser Tyr Asp Cys 865 870875 880 Val Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys Glu Phe Tyr Thr885 890 895 Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu Arg Leu LeuAsn 900 905 910 Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys Pro LysPhe Ser 915 920 925 Gly Val Glu Lys Ile Lys Thr Ile Gly Ser Thr Tyr MetAla Ala Thr 930 935 940 Gly Leu Ser Ala Val Pro Ser Gln Glu His Ser GlnGlu Pro Glu Arg 945 950 955 960 Gln Tyr Met His Ile Gly Thr Met Val GluPhe Ala Phe Ala Leu Val 965 970 975 Gly Lys Leu Asp Ala Ile Asn Lys HisSer Phe Asn Asp Phe Lys Leu 980 985 990 Arg Val Gly Ile Asn His Gly ProVal Ile Ala Gly Val Ile Gly Ala 995 1000 1005 Gln Lys Pro Gln Tyr AspIle Trp Gly Asn Thr Val Asn Val Ala 1010 1015 1020 Ser Arg Met Asp SerThr Gly Val Leu Asp Lys Ile Gln Val Thr 1025 1030 1035 Glu Glu Thr SerLeu Val Leu Gln Thr Leu Gly Tyr Thr Cys Thr 1040 1045 1050 Cys Arg GlyIle Ile Asn Val Lys Gly Lys Gly Asp Leu Lys Thr 1055 1060 1065 Tyr PheVal Asn Thr Glu Met Ser Arg Ser Leu Ser Gln Ser Asn 1070 1075 1080 ValAla Ser 1085 7 1090 PRT Rattus norvegicus 7 Met Arg Arg Arg Arg Tyr LeuArg Asp Arg Ala Glu Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Gly Gly GlyGlu Gly Leu Gln Arg Ser Arg Asp Trp Leu 20 25 30 Tyr Glu Ser Tyr Tyr CysMet Ser Gln Gln His Pro Leu Ile Val Phe 35 40 45 Leu Leu Leu Ile Val MetGly Ala Cys Leu Ala Leu Leu Ala Val Phe 50 55 60 Phe Ala Leu Gly Leu GluVal Glu Asp His Val Ala Phe Leu Ile Thr 65 70 75 80 Val Pro Thr Ala LeuAla Ile Phe Phe Ala Ile Phe Ile Leu Val Cys 85 90 95 Ile Glu Ser Val PheLys Lys Leu Leu Arg Val Phe Ser Leu Val Ile 100 105 110 Trp Ile Cys LeuVal Ala Met Gly Tyr Leu Phe Met Cys Phe Gly Gly 115 120 125 Thr Val SerAla Trp Asp Gln Val Ser Phe Phe Leu Phe Ile Ile Phe 130 135 140 Val ValTyr Thr Met Leu Pro Phe Asn Met Arg Asp Ala Ile Ile Ala 145 150 155 160Ser Ile Leu Thr Ser Ser Ser His Thr Ile Val Leu Ser Val Tyr Leu 165 170175 Ser Ala Thr Pro Gly Ala Lys Glu His Leu Phe Trp Gln Ile Leu Ala 180185 190 Asn Val Ile Ile Phe Ile Cys Gly Asn Leu Ala Gly Ala Tyr His Lys195 200 205 His Leu Met Glu Leu Ala Leu Gln Gln Thr Tyr Arg Asp Thr CysAsn 210 215 220 Cys Ile Lys Ser Arg Ile Lys Leu Glu Phe Glu Lys Arg GlnGln Glu 225 230 235 240 Arg Leu Leu Leu Ser Leu Leu Pro Ala His Ile AlaMet Glu Met Lys 245 250 255 Ala Glu Ile Ile Gln Arg Leu Gln Gly Pro LysAla Gly Gln Met Glu 260 265 270 Asn Thr Asn Asn Phe His Asn Leu Tyr ValLys Arg His Thr Asn Val 275 280 285 Ser Ile Leu Tyr Ala Asp Ile Val GlyPhe Thr Arg Leu Ala Ser Asp 290 295 300 Cys Ser Pro Gly Glu Leu Val HisMet Leu Asn Glu Leu Phe Gly Lys 305 310 315 320 Phe Asp Gln Ile Ala LysGlu Asn Glu Cys Met Arg Ile Lys Ile Leu 325 330 335 Gly Asp Cys Tyr TyrCys Val Ser Gly Leu Pro Ile Ser Leu Pro Asn 340 345 350 His Ala Lys AsnCys Val Lys Met Gly Leu Asp Met Cys Glu Ala Ile 355 360 365 Lys Lys ValArg Asp Ala Thr Gly Val Asp Ile Asn Met Arg Val Gly 370 375 380 Val HisSer Gly Asn Val Leu Cys Gly Val Ile Gly Leu Gln Lys Trp 385 390 395 400Gln Tyr Asp Val Trp Ser His Asp Val Thr Leu Ala Asn His Met Glu 405 410415 Ala Gly Gly Val Pro Gly Arg Val His Ile Ser Ser Val Thr Leu Glu 420425 430 His Leu Asn Gly Ala Tyr Lys Val Glu Glu Gly Asp Gly Glu Ile Arg435 440 445 Asp Pro Tyr Leu Lys Gln His Leu Val Lys Thr Tyr Phe Val IleAsn 450 455 460 Pro Lys Gly Glu Arg Arg Ser Pro Gln His Leu Phe Arg ProArg His 465 470 475 480 Thr Leu Asp Gly Ala Lys Met Arg Ala Ser Val ArgMet Thr Arg Tyr 485 490 495 Leu Glu Ser Trp Gly Ala Ala Lys Pro Phe AlaHis Leu His His Arg 500 505 510 Asp Ser Met Thr Thr Glu Asn Gly Lys IleSer Thr Thr Asp Val Pro 515 520 525 Met Gly Gln His Asn Phe Gln Asn ArgThr Leu Arg Thr Lys Ser Gln 530 535 540 Lys Lys Arg Phe Glu Glu Glu LeuAsn Glu Arg Met Ile Gln Ala Ile 545 550 555 560 Asp Gly Ile Asn Ala GlnLys Gln Trp Leu Lys Ser Glu Asp Ile Gln 565 570 575 Arg Ile Ser Leu LeuPhe Tyr Asn Lys Asn Ile Glu Lys Glu Tyr Arg 580 585 590 Ala Thr Ala LeuPro Ala Phe Lys Tyr Tyr Val Thr Cys Ala Cys Leu 595 600 605 Ile Phe LeuCys Ile Phe Ile Val Gln Ile Leu Val Leu Pro Lys Thr 610 615 620 Ser IleLeu Gly Phe Ser Phe Gly Ala Ala Phe Leu Ser Leu Ile Phe 625 630 635 640Ile Leu Phe Val Cys Phe Ala Gly Gln Leu Leu Gln Cys Ser Lys Lys 645 650655 Ala Ser Thr Ser Leu Met Trp Leu Leu Lys Ser Ser Gly Ile Ile Ala 660665 670 Asn Arg Pro Trp Pro Arg Ile Ser Leu Thr Ile Val Thr Thr Ala Ile675 680 685 Ile Leu Thr Met Ala Val Phe Asn Met Phe Phe Leu Ser Asn SerGlu 690 695 700 Glu Thr Thr Leu Pro Thr Ala Asn Thr Ser Asn Ala Asn ValSer Val 705 710 715 720 Pro Asp Asn Gln Ala Ser Ile Leu His Ala Arg AsnLeu Phe Phe Leu 725 730 735 Pro Tyr Phe Ile Tyr Ser Cys Ile Leu Gly LeuIle Ser Cys Ser Val 740 745 750 Phe Leu Arg Val Asn Tyr Glu Leu Lys MetLeu Ile Met Met Val Ala 755 760 765 Leu Val Gly Tyr Asn Thr Ile Leu LeuHis Thr His Ala His Val Leu 770 775 780 Asp Ala Tyr Ser Gln Val Leu PheGln Arg Pro Gly Ile Trp Lys Asp 785 790 795 800 Leu Lys Thr Met Gly SerVal Ser Leu Ser Ile Phe Phe Ile Thr Leu 805 810 815 Leu Val Leu Gly ArgGln Ser Glu Tyr Tyr Cys Arg Leu Asp Phe Leu 820 825 830 Trp Lys Asn LysPhe Lys Lys Glu Arg Glu Glu Ile Glu Thr Met Glu 835 840 845 Asn Leu AsnArg Val Leu Leu Glu Asn Val Leu Pro Ala His Val Ala 850 855 860 Glu HisPhe Leu Ala Arg Ser Leu Lys Asn Glu Glu Leu Tyr His Gln 865 870 875 880Ser Tyr Asp Cys Val Cys Val Met Phe Ala Ser Ile Pro Asp Phe Lys 885 890895 Glu Phe Tyr Thr Glu Ser Asp Val Asn Lys Glu Gly Leu Glu Cys Leu 900905 910 Arg Leu Leu Asn Glu Ile Ile Ala Asp Phe Asp Asp Leu Leu Ser Lys915 920 925 Pro Lys Phe Ser Gly Val Glu Lys Ile Lys Thr Ile Gly Ser ThrTyr 930 935 940 Met Ala Ala Thr Gly Leu Ser Ala Ile Pro Ser Gln Glu HisAla Gln 945 950 955 960 Glu Pro Glu Arg Gln Tyr Met His Ile Gly Thr MetVal Glu Phe Ala 965 970 975 Tyr Ala Leu Val Gly Lys Leu Asp Ala Ile AsnLys His Ser Phe Asn 980 985 990 Asp Phe Lys Leu Arg Val Gly Ile Asn HisGly Pro Val Ile Ala Gly 995 1000 1005 Val Ile Gly Ala Gln Lys Pro GlnTyr Asp Ile Trp Gly Asn Thr 1010 1015 1020 Val Asn Val Ala Ser Arg MetAsp Ser Thr Gly Val Leu Asp Lys 1025 1030 1035 Ile Gln Val Thr Glu GluThr Ser Leu Ile Leu Gln Thr Leu Gly 1040 1045 1050 Tyr Thr Cys Thr CysArg Gly Ile Ile Asn Val Lys Gly Lys Gly 1055 1060 1065 Asp Leu Lys ThrTyr Phe Val Asn Thr Glu Met Ser Arg Ser Leu 1070 1075 1080 Ser Gln SerAsn Leu Ala Ser 1085 1090

1. An isolated polynucleotide encoding a adenylate cyclase polypeptideand being selected from the group consisting of: a) a polynucleotideencoding a adenylate cyclase polypeptide comprising an amino acidsequence selected form the group consisting of: amino acid sequenceswhich are at least about 95% identical to the amino acid sequence shownin SEQ ID NO: 6; and the amino acid sequence shown in SEQ ID NO:
 6. b) apolynucleotide comprising the sequence of SEQ ID NO: 4 or 5; c) apolynucleotide which hybridizes under stringent conditions to apolynucleotide specified in (a) and (b); d) a polynucleotide thesequence of which deviates from the polynucleotide sequences specifiedin (a) to (c) due to the degeneration of the genetic code; and e) apolynucleotide which represents a fragment, derivative or allelicvariation of a polynucleotide sequence specified in (a to (d).
 2. Anexpression vector containing any polynucleotide of claim
 1. 3. A hostcell containing the expression vector of claim
 2. 4. A substantiallypurified adenylate cyclase polypeptide encoded by a polynucleotide ofclaim
 1. 5. A method for producing a adenylate cyclase polypeptide,wherein the method comprises the following steps: a) culturing the hostcell of claim 3 under conditions suitable for the expression of theadenylate cyclase polypeptide; and b) recovering the adenylate cyclasepolypeptide from the host cell culture.
 6. A method for detection of apolynucleotide encoding a adenylate cyclase polypeptide in a biologicalsample comprising the following steps: a) hybridizing any polynucleotideof claim 1 to a nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting said hybridizationcomplex.
 7. The method of claim 6, wherein before hybridization, thenucleic acid material of the biological sample is amplified.
 8. A methodfor the detection of a polynucleotide of claim 1 or a adenylate cyclasepolypeptide of claim 4 comprising the steps of: contacting a biologicalsample with a reagent which specifically interacts with thepolynucleotide or the adenylate cyclase polypeptide.
 9. A diagnostic kitfor conducting the method of any one of claims 6 to
 8. 10. A method ofscreening for agents which decrease the activity of a adenylate cyclase,comprising the steps of: contacting a test compound with any adenylatecyclase polypeptide encoded by any polynucleotide of claim 1; detectingbinding of the test compound to the adenylate cyclase polypeptide,wherein a test compound which binds to the polypeptide is identified asa potential therapeutic agent for decreasing the activity of a adenylatecyclase.
 11. A method of screening for agents which regulate theactivity of a adenylate cyclase, comprising the steps of: contacting atest compound with a adenylate cyclase polypeptide encoded by anypolynucleotide of claim 1; and detecting a adenylate cyclase activity ofthe polypeptide, wherein a test compound which increases the adenylatecyclase activity is identified as a potential therapeutic agent forincreasing the activity of the adenylate cyclase, and wherein a testcompound which decreases the adenylate cyclase activity of thepolypeptide is identified as a potential therapeutic agent fordecreasing the activity of the adenylate cyclase.
 12. A method ofscreening for agents which decrease the activity of a adenylate cyclase,comprising the steps of: contacting a test compound with anypolynucleotide of claim 1 and detecting binding of the test compound tothe polynucleotide, wherein a test compound which binds to thepolynucleotide is identified as a potential therapeutic agent fordecreasing the activity of adenylate cyclase.
 13. A method of reducingthe activity of adenylate cyclase, comprising the steps of: contacting acell with a reagent which specifically binds to any polynucleotide ofclaim 1 or any adenylate cyclase polypeptide of claim 4, whereby theactivity of adenylate cyclase is reduced.
 14. A reagent that modulatesthe activity of a adenylate cyclase polypeptide or a polynucleotidewherein said reagent is identified by the method of any of the claim 10to
 12. 15. A pharmaceutical composition, comprising: the expressionvector of claim 2 or the reagent of claim 14 and a pharmaceuticallyacceptable carrier.
 16. Use of the expression vector of claim 2 or thereagent of claim 14 in the preparation of a medicament for modulatingthe activity of a adenylate cyclase in a disease.
 17. Use of claim 16wherein the disease is peripheral or central nervous system disorders,disorders of the genito-urinary system, obesity, COPD or diabetes.
 18. AcDNA encoding a polypeptide comprising the amino acid sequence shown inSEQ ID NO:2 or
 4. 19. The cDNA of claim 18 which comprises SEQ ID NO: 4or
 5. 20. The cDNA of claim 18 which consists of SEQ ID NO: 4 or
 5. 21.An expression vector comprising a polynucleotide which encodes apolypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.22. The expression vector of claim 21 wherein the polynucleotideconsists of SEQ ID NO: 4 or
 5. 23. A host cell comprising an expressionvector which encodes a polypeptide comprising the amino acid sequenceshown in SEQ ID NO:
 6. 24. The host cell of claim 23 wherein thepolynucleotide consists of SEQ ID NO: 4 or
 5. 25. A purified polypeptidecomprising the amino acid sequence shown in SEQ D NO:
 6. 26. Thepurified polypeptide of claim 25 which consists of the amino acidsequence shown in SEQ ID NO:
 6. 27. A fusion protein comprising apolypeptide having the amino acid sequence shown in SEQ ID NO:
 6. 28. Amethod of producing a polypeptide comprising the amino acid sequenceshown in SEQ ID NO: 6, comprising the steps of: culturing a host cellcomprising an expression vector which encodes the polypeptide underconditions whereby the polypeptide is expressed; and isolating thepolypeptide.
 29. The method of claim 28 wherein the expression vectorcomprises SEQ ID NO: 4 or S.
 30. A method of detecting a coding sequencefor a polypeptide comprising the amino acid sequence shown in SEQ ID NO:6, comprising the steps of: hybridizing a polynucleotide comprising 11contiguous nucleotides of SEQ ID NO: 4 or 5 to nucleic acid material ofa biological sample, thereby forming a hybridization complex; anddetecting the hybridization complex.
 31. The method of claim 30 furthercomprising the step of amplifying the nucleic acid material before thestep of hybridizing.
 32. A kit for detecting a coding-sequence for apolypeptide comprising the amino acid sequence shown in SEQ ID NO: 6,comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQID NO: 4 or 5; and instructions for the method of claim
 30. 33. A methodof detecting a polypeptide comprising the amino acid sequence shown inSEQ ID NO: 6, comprising the steps of: contacting a biological samplewith a reagent that specifically binds to the polypeptide to form areagent-polypeptide complex; and detecting the reagent-polypeptidecomplex.
 34. The method of claim 33 wherein the reagent is an antibody.35. A kit for detecting a polypeptide comprising the amino acid sequenceshown in SEQ ID NO: 6, comprising: an antibody which specifically bindsto the polypeptide; and instructions for the method of claim
 33. 36. Amethod of screening for agents which can modulate the activity of ahuman adenylate cyclase, comprising the steps of: contacting a testcompound with a polypeptide comprising an amino acid sequence selectedfrom the group consisting of (1) amino acid sequences which are at leastabout 95% identical to the amino acid sequence shown in SEQ ID NO: 6 and(2) the amino acid sequence shown in SEQ ID NO: 6; and detecting bindingof the test compound to the polypeptide, wherein a test compound whichbinds to the polypeptide is identified as a potential agent forregulating activity of the human adenylate cyclase.
 37. The method ofclaim 36 wherein the step of contacting is in a cell.
 38. The method ofclaim 36 wherein the cell is in vitro.
 39. The method of claim 36wherein the step of contacting is in a cell-free system.
 40. The methodof claim 36 wherein the polypeptide comprises a detectable label. 41.The method of claim 36 wherein the test compound comprises a detectablelabel.
 42. The method of claim 36 wherein the test compound displaces alabeled ligand which is bound to the polypeptide.
 43. The method ofclaim 36 wherein the polypeptide is bound to a solid support.
 44. Themethod of claim 36 wherein the test compound is bound to a solidsupport.
 45. A method of screening for agents which modulate an activityof a human adenylate cyclase, comprising the steps of: contacting a testcompound with a polypeptide comprising an amino acid sequence selectedfrom the group consisting of: (1) amino acid sequences which are atleast about 95% identical to the amino acid sequence shown in SEQ ID NO:6 and (2) the amino acid sequence shown in SEQ ID NO: 6; and detectingan activity of the polypeptide, wherein a test compound which increasesthe activity of the polypeptide is identified as a potential agent forincreasing the activity of the human adenylate cyclase, and wherein atest compound which decreases the activity of the polypeptide isidentified as a potential agent for decreasing the activity of the humanadenylate cyclase.
 46. The method of claim 45 wherein the step ofcontacting is in a cell.
 47. The method of claim 45 wherein the cell isin vitro.
 48. The method of claim 45 wherein the step of contacting isin a cell-free system.
 49. A method of screening for agents whichmodulate an activity of a human adenylate cyclase, comprising the stepsof: contacting a test compound with a product encoded by apolynucleotide which comprises the nucleotide sequence shown in SEQ IDNO: 4 or 5; and detecting binding of the test compound to the product,wherein a test compound which binds to the product is identified as apotential agent for regulating the activity of the human adenylatecyclase.
 50. The method of claim 49 wherein the product is apolypeptide.
 51. The method of claim 49 wherein the product is RNA. 52.A method of reducing activity of a human adenylate cyclase, comprisingthe step of: contacting a cell with a reagent which specifically bindsto a product encoded by a polynucleotide comprising the nucleotidesequence shown in SEQ ID NO: 4 or 5, whereby the activity of a humanadenylate cyclase is reduced.
 53. The method of claim 52 wherein theproduct is a polypeptide.
 54. The method of claim 53 wherein the reagentis an antibody.
 55. The method of claim 52 wherein the product is RNA.56. The method of claim 55 wherein the reagent is an antisenseoligonucleotide.
 57. The method of claim 56 wherein the reagent is aribozyme.
 58. The method of claim 52 wherein the cell is in vitro. 59.The method of claim 52 wherein the cell is in vivo.
 60. A pharmaceuticalcomposition, comprising: a reagent which specifically binds to apolypeptide comprising the amino acid sequence shown in SEQ ID NO: 6;and a pharmaceutically acceptable carrier.
 61. The pharmaceuticalcomposition of claim 60 wherein the reagent is an antibody.
 62. Apharmaceutical composition, comprising: a reagent which specificallybinds to a product of a polynucleotide comprising the nucleotidesequence shown in SEQ ID NO: 4 or 5; and a pharmaceutically acceptablecarrier.
 63. The pharmaceutical composition of claim 62 wherein thereagent is a ribozyme.
 64. The pharmaceutical composition of claim 62wherein the reagent is an antisense oligonucleotide.
 65. Thepharmaceutical composition of claim 62 wherein the reagent is anantibody.
 66. A pharmaceutical composition, comprising: an expressionvector encoding a polypeptide comprising the amino acid sequence shownin SEQ ID NO: 6; and a pharmaceutically acceptable carrier.
 67. Thepharmaceutical composition of claim 66 wherein the expression vectorcomprises SEQ ID NO: 4 or
 5. 68. A method of treating a adenylatecyclase dysfunction related disease, wherein the disease is selectedfrom peripheral or central nervous system disorders, disorders of thegenito-urinary system, obesity, COPD or diabetes comprising the step of:administering to a patient in need thereof a therapeutically effectivedose of a reagent that modulates a function of a human adenylatecyclase, whereby symptoms of the adenylate cyclase dysfunction relateddisease are ameliorated.
 69. The method of claim 68 wherein the reagentis identified by the method of claim
 36. 70. The method of claim 68wherein the reagent is identified by the method of claim
 45. 71. Themethod of claim 68 wherein the reagent is identified by the method ofclaim 49.